Compositions and methods for modulating angiogenesis

ABSTRACT

The invention generally features compositions and methods that are useful for modulating angiogenesis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.12/309,788, filed Mar. 15, 2010, which is the U.S. national phase,pursuant to 35 U.S.C. §371, of PCT international application Ser. No.PCT/US2007/017085, filed Jul. 30, 2007, designating the United Statesand published in English on Jan. 31, 2008 as publication WO 2008/014008A2, which claims priority to U.S. provisional patent application Ser.No. 60/834,252, filed Jul. 28, 2006. The entire disclosure of theaforementioned patent applications are incorporated herein by thisreference.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are 21-23 nucleotide RNA molecules that regulate thestability or translational efficiency of target mRNAs. miRNAs havediverse functions including the regulation of cellular differentiation,proliferation, and apoptosis. Although strict tissue- anddevelopmental-stage-specific expression is critical for appropriatemiRNA function, few mammalian transcription factors that regulate miRNAshave been identified.

Angiogenesis refers to the formation of new blood vessels, and isessential to proper embryonic development and growth, and tissue repair.Angiogenesis is also essential to many pathological conditions,including neoplasia, coronary artery disease, vascular disease,rheumatoid arthritis, psoriasis, and diabetic retinopathy.

Accordingly, improved compositions and methods for the prevention ofangiogenesis and angiogenesis related diseases and disorders arerequired.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions andmethods for modulating angiogenesis in a subject.

In one aspect, the invention generally features a method of reducingangiogenesis (e.g., by at least about 5%, 10%, 25%, 50%, 75%, or 100%),the method comprising contacting a cell (e.g., a human cell in vitro orin vivo) with an effective amount of an inhibitory nucleic acid molecule(e.g., antisense or siRNA) complementary to at least a portion of amicroRNA nucleic acid molecule of the mir-17-92 cluster (e.g., any oneor more of mir-17-5p, mir-18a, mir-19a, mir-20a, mir-19b-1, andmir-92-1), thereby reducing angiogenesis. In one embodiment, theinhibitory nucleic acid molecule decreases the expression of themicroRNA in the cell. In another embodiment, the cell is present in atissue or organ. In yet another embodiment, the cell is a neoplasticcell or an ocular cell. In another embodiment, the cell is contactedwith at least two, three, four, five or six inhibitory nucleic acidmolecules. In yet another embodiment, the inhibitory nucleic acidmolecules reduce the expression of mir-19 and mir-18. In yet anotherembodiment, the method increases expression of Tsp1 or CTGF relative toa reference (e.g., the level of Tsp1 or CTGF expression in the cell,tissue, or organ prior to treatment or the level present in acorresponding healthy or neoplastic control cell, tissue, or organ). Inyet another embodiment, the TSR protein is selected from the groupconsisting of thrombospondin type 1 repeats (TSR): spondin-1(f-spondin), thrombospondin-1, ADAMTS2, WISP2, thrombospondinrepeat-containing protein 1, clusterin, connective tissue growth factor(CTGF), secreted acidic cysteine rich glycoprotein (SPARC), ADAMTS12,thrombospondin type I domain-containing protein 6. In yet anotherembodiment, the TSR protein is selected from the group consisting ofthrombospondin type 1 repeats (TSR): CTGF, THSD3, ADAMTS18, ADAMTS12,THBS 1, THSD1, ADAMTS1, ADAMTS6, WISP2, and BAI3. In still anotherembodiment, the antisense nucleic acid molecule comprises a nucleobasesequence having at least 85%, 90%, or 95% identity to a sequenceselected from any one or more of the following:

miR-17-5p: ACUACCUGCACuGUAAGCACUUUG; mir-18a: UAUGUGCACUAGAUGCACCUUA;mir-19a: UCAGUUUUGCAUAGAUUUGCACA; mir-19b: UCAGUUUUGCAUGGAUUUGCACA;mir-20a: CUACCUGCACUAUAAGCACUUUA; and mir-92-1: CAGGCCGGGACAAGUGCAAUA.In other embodiments, the inhibitory nucleic acid molecule consistssubstantially of any one of those sequences.

In another aspect, the invention provides a method for increasing theexpression of a TSR protein in a cell, the method comprising contactingthe cell with an effective amount of an inhibitory nucleic acid moleculecomplementary to at least a portion of a microRNA nucleic acid moleculeof the mir-17-92 cluster, thereby increasing the expression of a TSRprotein. In one embodiment, the contact increases expression of athrombospondin type 1 repeat (TSR) protein (e.g., Tsp1 or CTGF) relativeto a reference.

In yet another aspect, the invention features a method of treating anapoptosis resistant neoplasm or chemo-resistant neoplasm in a subject.The method involves identifying a subject as having an apoptosisresistant neoplasm; and administering to the subject an effective amountof an inhibitory nucleic acid molecule complementary to at least aportion of a microRNA of the mir-17-92 cluster.

In yet another aspect, the invention features a method of treating orpreventing a neoplasm in a subject in need thereof, the method involvingidentifying a neoplasm having an increase in the expression of a TSRprotein; and administering to the subject an effective amount of aninhibitory nucleic acid molecule complementary to at least a portion ofa microRNA of the mir-17-92 cluster.

In still another aspect, the invention features a method of treating anocular disease characterized by increased angiogenesis in a subject, themethod comprising administering to the subject an effective amount of aninhibitory nucleic acid molecule complementary to at least a portion ofa microRNA of the mir-17-92 cluster. In one embodiment, the oculardisease is macular degeneration, age-related macular degeneration,choroidal neovascularization, or diabetic retinopathy.

In yet another aspect, the invention provides a method of characterizinga tumor as amenable to treatment with the method of previous aspect, themethod comprising assaying the expression of a microRNA encoded by themiR-17-92 cluster.

In another aspect, the invention provides a method of characterizing atumor as amenable to treatment, the method comprising assaying theexpression of a TSR protein. In one embodiment, the method detects anincrease or a decrease in the expression of a TSR protein relative to areference.

In yet another aspect, the invention features a method of selecting atreatment for a subject having a neoplasm, the method involvingdetecting an increase the expression of a microRNA encoded by amir-17-92 cluster or a TSR protein; and identifying the patient ashaving a neoplasm amenable to treatment with an inhibitory nucleic acidmolecule that reduces the expression of a microRNA of the miR-17-92cluster. In one embodiment, the method detects an increase in theexpression of a microRNA and a TSR protein.

In another aspect, the invention provides a method of monitoring thetreatment of a subject having a neoplasia, the method involving assayingthe expression of a TSR protein in a cell of the subject and detectingan increase or a decrease in the expression of a TSR protein relative toa reference. In one embodiment, an increase in the expression of the TSRprotein indicates that the treatment is beneficial.

In yet another aspect, the invention features an isolated nucleic acidmolecule having at least 85%, 90%, 95% or greater nucleic acid sequenceidentity to a microRNA encoded by the miR-17-92 cluster, whereinexpression of the nucleic acid molecule in a cell enhances angiogenesis.In one embodiment, the nucleic acid molecule has at least 85%, 90%, 95%or greater sequence identity to (human) a microRNA selected from thegroup consisting of miR-17-92, miR-19, and miR-18. In other embodiments,the nucleic acid molecule comprises at least one or more modifications,such as a non-natural internucleotide linkage, modified backbone,substituted sugar moiety, or cholesterol conjugation.

In another aspect, the invention provides an expression vector (e.g., aretroviral, adenoviral, adeno-associated viral, or lentiviral vector)encoding a nucleic acid molecule of any previous aspect. In oneembodiment, the vector contains a promoter suitable for expression in amammalian cell, wherein the promoter is operably linked to the nucleicacid molecule.

In another aspect, the invention provides a cell (e.g., a human cell invivo or in vitro) containing the vector of the previous aspect or anucleic acid molecule of any previous aspect. In one embodiment, thecell is a neoplastic colonocyte cell in vivo. In another embodiment, thecell is apoptosis-resistant or chemotherapy resistant.

In yet another aspect, the invention features method of enhancingangiogenesis, the method comprising contacting the cell with aneffective amount of a nucleic acid molecule comprising at least aportion of a microRNA nucleic acid molecule of the mir-17-92 cluster. Inone embodiment, the cell further expresses one or more of athrombospondin family protein.

In another aspect, the invention provides a method of identifying anagent that reduces angiogenesis, the method involving contacting a cellthat expresses a TSR selected from the group consisting of: Connectivetissue growth factor (CTGF), thrombospondin, type I domain containing 3isoform 3 (THSD3), A disintegrin and metalloproteinase with Tsp motifs18 (ADAMTS18), A disintegrin and metalloproteinase with Tsp motifs 12(ADAMTS12), Thrombospondin 1 (THBS1), Thrombospondin, type 1 domaincontaining 1 (THSD1), A disintegrin and metalloproteinase with Tspmotifs 1 (ADAMTS1), A disintegrin and metalloproteinase with Tsp motifs6 (ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3) and a microRNA of themir-17-92 cluster with a test agent; and detecting an increase in thelevel of TSR expression in the cell contacted by the agent with thelevel present in a control cell, wherein the increase in TSR expressionidentifying the agent as reducing angiogenesis.

In yet another aspect, the invention provides a method of identifying anagent that treats or prevents an apoptosis resistant neoplasm, themethod comprising contacting a cell that expresses a microRNA of themir-17-92 cluster with an agent, and detecting a reduction in the levelof microRNA expression in the cell contacted by the agent with the levelof expression in a control cell, wherein an agent that decreasesmicroRNA expression thereby treats or prevents a neoplasm. In variousembodiments, the cell further expresses reduced levels of a TSR proteinthat is any one or more of Connective tissue growth factor (CTGF),thrombospondin, type I domain containing 3 isoform 3 (THSD3), Adisintegrin and metalloproteinase with Tsp motifs 18 (ADAMTS18), Adisintegrin and metalloproteinase with Tsp motifs 12 (ADAMTS12),Thrombospondin 1 (THBS1), Thrombospondin, type 1 domain containing 1(THSD1), A disintegrin and metalloproteinase with Tsp motifs 1(ADAMTS1), A disintegrin and metalloproteinase with Tsp motifs 6(ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3) relative to a referencecell. In one embodiment, the decrease in expression is by at least about5%, 10%, 15%, 25%, 30%, 50%, 75% or more.

In another aspect, the invention provides a method for diagnosing asubject as having or having a propensity to develop an apoptosisresistant neoplasia, the method involving measuring the level of a TSRprotein selected from any one or more of Connective tissue growth factor(CTGF), thrombospondin, type I domain containing 3 isoform 3 (THSD3), Adisintegrin and metalloproteinase with Tsp motifs 18 (ADAMTS18), Adisintegrin and metalloproteinase with Tsp motifs 12 (ADAMTS12),Thrombospondin 1 (THBS1), Thrombospondin, type 1 domain containing 1(THSD1), A disintegrin and metalloproteinase with Tsp motifs 1(ADAMTS1), A disintegrin and metalloproteinase with Tsp motifs 6(ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3) in a biological samplefrom the subject; and comparing the level of the TSR protein in thesubject to the level present in a control subject, wherein a reducedlevel of TSR protein indicates the subject has or has a propensity todevelop an apoptosis resistant neoplasia.

In another aspect, the invention provides a method for diagnosing asubject as having or having a propensity to develop an apoptosisresistant neoplasia, the method involving measuring the level of amir17-92 encoded microRNA in a biological sample derived from a subject;and detecting an increased level of the microRNA relative to the levelpresent in a control sample, wherein a, wherein an increase in the levelof the mir-17-92 encoded microRNA indicates the subject has or has apropensity to develop a an apoptosis resistant neoplasia. In oneembodiment, the level of mir-17-92 is detected in a microarray assay, animmunoassay, or a radioassay. In another embodiment, the methodcomprises measuring the level of nucleic acid molecule or polypeptide.

In yet another aspect, the invention features a pharmaceuticalcomposition or kit for treating an apoptosis resistant neoplasm in asubject comprising an effective amount of an inhibitory nucleic acidmolecule that is complementary to at least a fragment of mir-17-92 in apharmaceutically acceptable excipient.

In various embodiments of any of the previous aspects, the TSR proteinis Connective tissue growth factor (CTGF), thrombospondin, type I domaincontaining 3 isoform 3 (THSD3), A disintegrin and metalloproteinase withTsp motifs 18 (ADAMTS18), A disintegrin and metalloproteinase with Tspmotifs 12 (ADAMTS12), Thrombospondin 1 (THBS1), Thrombospondin, type 1domain containing 1 (THSD1), A disintegrin and metalloproteinase withTsp motifs 1 (ADAMTS1), A disintegrin and metalloproteinase with Tspmotifs 6 (ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2),and Brain-specific angiogenesis inhibitor 3 (BAI3). In otherembodiments, the reference is the level of Tsp1 or CTGF expression inthe cell prior to treatment or the level present in a correspondingneoplastic control cell. In still other embodiments, the inhibitorynucleic acid molecule is an antisense and siRNA nucleic acid molecule.In yet other embodiments, the antisense nucleic acid molecule comprisesa nucleobase sequence having at least 85%, 90% or 95% identity to asequence selected from any one or more (e.g., two, three, four, five orsix) of the following:

miR-17-5p: ACUACCUGCACuGUAAGCACUUUG; mir-18a: UAUGUGCACUAGAUGCACCUUA;mir-19a: UCAGUUUUGCAUAGAUUUGCACA; mir-19b: UCAGUUUUGCAUGGAUUUGCACA;mir-20a: CUACCUGCACUAUAAGCACUUUA; and mir-92-1: CAGGCCGGGACAAGUGCAAUA.In still other embodiments of the above aspects, the cell is aneoplastic cell or an ocular cell. In other embodiments, the methodincreases expression of Tsp1 or CTGF relative to a reference. In yetother embodiments, the reference is the level of Tsp1 or CTGF expressionin the cell prior to treatment or the level present in a correspondingneoplastic control cell. In other embodiments, the inhibitory nucleicacid molecule is an antisense and siRNA nucleic acid molecule. In stillother embodiments, the cell is contacted with at least two, three, four,five, or six inhibitory nucleic acid molecules. In other embodiments,the inhibitory nucleic acid molecules reduce the expression of mir-19and mir-18. In yet other embodiments of the previous aspects, the one ormore inhibitory nucleic acid molecules are administered concurrently orwithin 14 days of each other in amounts sufficient to inhibit the growthof the apoptosis resistant neoplasm. In other embodiments of the aboveaspects, the neoplasm is selected from the group consisting of lungcancer, breast cancer, cervical cancer, colon cancer, gastric cancer,kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer,pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer,testicular cancer, and uterine cancer. In still other embodiments of theabove aspects, the inhibitory nucleic acid molecule is administered atin an amount between about 100 to 300 mg/m2/day. In still otherembodiments, the inhibitory nucleic acid molecule is administeredsystemically or locally to a subject. In still other embodiments, themethod of administration targets the tumor and/or the tumormicroenvironment (e.g., stromal cells, fibroblasts, endothelial cells,inflammatory cells, smooth muscle cells, and pericytes).

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “agent” is meant a polypeptide, polynucleotide, or fragment, oranalog thereof, small molecule, or other biologically active molecule.

By “alteration” is meant a change (increase or decrease) in theexpression levels of a gene or polypeptide as detected by standard artknown methods such as those described above. As used herein, analteration includes a 10% change in expression levels, preferably a 25%change, more preferably a 40% change, and most preferably a 50% orgreater change in expression levels.

By “antisense molecule” is meant a non-enzymatic nucleic acid moleculeor analog or variant thereof that binds to a target nucleic acidmolecule sequence by means of complementary base pairing, such as anRNA-RNA or RNA-DNA interactions and alters the expression of the targetsequence. Typically, antisense molecules are complementary to a targetsequence along a single contiguous sequence of the antisense molecule.In certain embodiments, an antisense molecule can bind to substrate suchthat the substrate molecule forms a loop, and/or an antisense moleculecan bind such that the antisense molecule forms a loop. Thus, theantisense molecule can be complementary to two (or even more)non-contiguous substrate sequences or two (or even more) non-contiguoussequence portions of a target sequence.

The phrase “in combination with” is intended to refer to all forms ofadministration that provide an inhibitory nucleic acid molecule togetherwith a second agent, such as a second inhibitory nucleic acid moleculeor a chemotherapeutic agent, where the two are administered concurrentlyor sequentially in any order.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

By “complementary” is meant capable of pairing to form a double-strandednucleic acid molecule or portion thereof. In one embodiment, anantisense molecule is in large part complementary to a target sequence.The complementarity need not be perfect, but may include mismatches at1, 2, 3, or more nucleotides.

By “control” is meant a standard or reference condition.

By “corresponds” is meant comprising at least a fragment of adouble-stranded gene, such that a strand of the double-strandedinhibitory nucleic acid molecule is capable of binding to acomplementary strand of the gene.

By “decreases” is meant a reduction by at least about 5% relative to areference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, oreven by as much as 75%, 85%, 95% or more.

By “an effective amount” is meant the amount of an agent required toameliorate the symptoms of a disease or slow, stabilize, prevent, orreduce the severity of the pathology in a subject relative to anuntreated subject. The effective amount of active agent(s) used topractice the present invention for therapeutic treatment of a neoplasiavaries depending upon the manner of administration, the age, bodyweight, and general health of the subject. Ultimately, the attendingphysician or veterinarian will decide the appropriate amount and dosageregimen. Such amount is referred to as an “effective” amount.

By “fragment” is meant a portion (e.g., at least 5, 10, 25, 50, 100,125, 150, 200, 250, 300, 350, 400, or 500 amino acids or nucleic acids)of a protein or nucleic acid molecule that is substantially identical toa reference protein or nucleic acid and retains the biological activityof the reference.

A “host cell” is any prokaryotic or eukaryotic cell that contains eithera cloning vector or an expression vector. This term also includes thoseprokaryotic or eukaryotic cells that have been genetically engineered tocontain the cloned gene(s) in the chromosome or genome of the host cell.

By “inhibitory nucleic acid molecule” is meant a single stranded ordouble-stranded RNA, siRNA (short interfering RNA), shRNA (short hairpinRNA), or antisense RNA, or a portion thereof, or an analog or mimeticthereof, that when administered to a mammalian cell results in adecrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in theexpression (e.g., transcription or translation) of a target sequence.Typically, a nucleic acid inhibitor comprises or corresponds to at leasta portion of a target nucleic acid molecule, or an ortholog thereof, orcomprises at least a portion of the complementary strand of a targetnucleic acid molecule.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

The term “microarray” is meant to include a collection of nucleic acidmolecules or polypeptides from one or more organisms arranged on a solidsupport (for example, a chip, plate, or bead).

By “miR-17-92 cluster” is meant the cluster of microRNAs located onchromosome 13 that encodes miRs-17-5p, 18a, 19a, 20a, 19-b1, and 92-1.The sequence of the primary transcript containing all the microRNAspresent in the cluster is provided at GenBank Accession No. AB 176708.

By “mir-17-5p” is meant a microRNA comprising or having at least 85%identity to the nucleic acid sequence provided at Genbank Accession No.AF480529. By “mir-18a” is meant a microRNA comprising or having at least85% identity to the nucleic acid sequence provided at GenBank AccessionNo. AJ421736.

By “mir-19a” is meant a microRNA comprising or having at least 85%identity to the nucleic acid sequence provided at GenBank Accession No.AJ421737.

By “mir-20a” is meant a microRNA comprising or having at least 85%identity to the nucleic acid sequence provided at Genbank Accession No.AJ421738.

By “mir-19b-1” is meant a microRNA comprising or having at least 85%identity to the nucleic acid sequence provided at Genbank Accession No.AJ421739.

By “mir-92-1 is meant a microRNA comprising or having at least 85%identity to the nucleic acid sequence provided at Genbank Accession No.AF480530.

By “modification” is meant any biochemical or other synthetic alterationof a nucleotide, amino acid, or other agent relative to a naturallyoccurring reference agent.

By “neoplasia” is meant any disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. For example, cancer is a neoplasia. Examples ofcancers include, without limitation, leukemias (e.g., acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).Lymphoproliferative disorders are also considered to be proliferativediseases.

By “nucleic acid” is meant an oligomer or polymer of ribonucleic acid ordeoxyribonucleic acid, or analog thereof. This term includes oligomersconsisting of naturally occurring bases, sugars, and intersugar(backbone) linkages as well as oligomers having non-naturally occurringportions which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofproperties such as, for example, enhanced stability in the presence ofnucleases.

By “obtaining” as in “obtaining the inhibitory nucleic acid molecule” ismeant synthesizing, purchasing, or otherwise acquiring the inhibitorynucleic acid molecule.

By “operably linked” is meant that a first polynucleotide is positionedadjacent to a second polynucleotide that directs transcription of thefirst polynucleotide when appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the second polynucleotide.

By “positioned for expression” is meant that the polynucleotide of theinvention (e.g., a DNA molecule) is positioned adjacent to a DNAsequence that directs transcription and translation of the sequence(i.e., facilitates the production of, for example, a recombinantmicroRNA molecule described herein).

By “portion” is meant a fragment of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides.

By “reference” is meant a standard or control condition.

By “reporter gene” is meant a gene encoding a polypeptide whoseexpression may be assayed; such polypeptides include, withoutlimitation, glucuronidase (GUS), luciferase, chloramphenicoltransacetylase (CAT), and beta-galactosidase.

The term “siRNA” refers to small interfering RNA; a siRNA is a doublestranded RNA that “corresponds” to or is complementary to a reference ortarget gene sequence. This matching need not be perfect so long as eachstrand of the siRNA is capable of binding to at least a portion of thetarget sequence. SiRNA can be used to inhibit gene expression, see forexample Bass, 2001, Nature, 411, 428 429; Elbashir et al., 2001, Nature,411, 494 498; and Zamore et al., Cell 101:25-33 (2000).

The term “subject” is intended to include vertebrates, preferably amammal. Mammals include, but are not limited to, humans.

The term “pharmaceutically-acceptable excipient” as used herein meansone or more compatible solid or liquid filler, diluents or encapsulatingsubstances that are suitable for administration into a human.

By “specifically binds” is meant a molecule (e.g., peptide,polynucleotide) that recognizes and binds a protein or nucleic acidmolecule of the invention, but which does not substantially recognizeand bind other molecules in a sample, for example, a biological sample,which naturally includes a protein of the invention.

By “substantially identical” is meant a protein or nucleic acid moleculeexhibiting at least 50% identity to a reference amino acid sequence (forexample, any one of the amino acid sequences described herein) ornucleic acid sequence (for example, any one of the nucleic acidsequences described herein). Preferably, such a sequence is at least60%, more preferably 80% or 85%, and still more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e-3 and e-100 indicating a closely related sequence.

By “TSR protein” is meant a thrombospondin type 1 repeats (TSR)containing protein or fragment thereof that modulates angiogenesis.Exemplary proteins include CTGF, THSD3, ADAMTS18, ADAMTS12, THBS1,THSD1, ADAMTS1, ADAMTS6, WISP2, and BAI3, and other proteins having atleast 80%, 85%, 90%, 95% or greater amino acid sequence identity to sucha protein.

By “targets” is meant alters the biological activity of a targetpolypeptide or nucleic acid molecule.

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, apolynucleotide molecule encoding (as used herein) a protein of theinvention.

By “vector” is meant a nucleic acid molecule, for example, a plasmid,cosmid, or bacteriophage, that is capable of replication in a host cell.In one embodiment, a vector is an expression vector that is a nucleicacid construct, generated recombinantly or synthetically, bearing aseries of specified nucleic acid elements that enable transcription of anucleic acid molecule in a host cell. Typically, expression is placedunder the control of certain regulatory elements, including constitutiveor inducible promoters, tissue-preferred regulatory elements, andenhancers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show growth properties of RasGfp versus RasGfpMyc p53-nullcolonocytes in vivo and in vitro. FIG. 1A is a graph showing the resultsof a cell accumulation assay performed on RasGfp and RasGfpMyccolonocytes. The number of viable cells in triplicate plates wasassessed using the water-soluble tetrazolium-1 (WST) assay. The insetpanel shows Myc overexpression detected by immunoblotting using mouseβ-actin as a loading control. FIG. 1B is a graph showing the averageweights of subcutaneous tumors formed by RasGfp (bars 1, 3, 4),RasGfpMyc (bar 2) and RasGfpMycER (bars 5, 6) colonocytes. Whereindicated, tumor-bearing animals were treated with 4-hydroxytamoxifen(4OHT) (‘with 4OHT’) or left untreated (‘w/o 4OHT’). Error bars in FIGS.1A and 1B refer to standard deviation (s.d.). FIG. 1C shows the resultsof immunostaining as a comparative analysis of RasGfp and RasGfp Myctumors. The left pinels are hematoxylin and eosin staining (H&E).Perfused blood vessels contain numerous red blood cells. The centerpanels show staining of endothelial cells with lectin (brown). The rightpanels show staining of lymphatic vessels with an antibody againstLYVE-1 (brown). A large vessel in the RasGfpMyc LYVE-1 image localizesin the surrounding adipose tissue. The inset in the right panels depictsstaining of normal ileum.

FIGS. 2A-2G show expression of pro- and anti-angiogenic factors inRasGfp and RasGfpMyc carcinomas. FIG. 2A is an immunoblot that showslack of detectable HIF1a in RasGfp and RasGfpMyc tumor lysates. Twoindependent tumors of each type (T1 and T2) were assayed. Mouseembryonic stem (ES) cells cultured in the presence or absence of hypoxiamimetic desferrioxamine (DFX) were used for comparison. FIG. 2B showsELISA-based quantification of VEGF A in the same neoplasms shown in FIG.2A. FIG. 2C is a graph depicting the results of real-time RT-PCRanalysis of thrombospondin-1 (thbs1) and connective tissue growth factor(CTGF) gene expression in the same neoplasms shown in FIG. 2A. Errorbars refer to standard deviation (s.d.). FIG. 2D shows immunoblottinganalysis of Tsp1 and CTGF expression levels. Cultured cells were usedfor CTGF detection. FIG. 2E shows immunoblotting analysis of CTGFexpression levels in mass cultures of RasGfpMycER cells treated with4OHT for the indicated number of hours. Whole-cell lysates andconditioned medium were analyzed. RasGfp and RasGfpMyc cells were usedfor comparison. FIG. 2F shows immunoblotting analysis as carried out inFIG. 2E performed on three single-cell RasGfpMycER clones. In theanalysis of conditioned medium, thrombospondin type 1 repeat (TSR)protein levels were normalized to cell numbers. FIG. 2G showsimmunoblotting analysis of Tsp1 and CTGF expression levels inRasGfpMycER clone 3. Assayed cells were initially treated with 4OHT for72 hours (shown on the left) and then deprived of 4OHT for additional 72hours (shown on the right). The trimeric form of Tsp1 was predominantlyexpressed in these lysates.

FIGS. 3A-3F show miR-17-92 and TSR protein expression in RasGfp andRasGfpMyc cells. FIG. 3A shows real-time RT-PCR analysis of themiR-17-92 primary transcript. The same tumors were tested here as inFIG. 2. The upper panel depicts PCR products quantified in the bargraphs shown in the lower panel. Error bars refer to s.d. FIG. 3B showsRNA blot analysis of four RasGfp and four RasGfpMyc tumors. The miR-18probe detects both pre-miR-18 and mature miR-18 species. U6 RNA was usedas a loading control. Numbers below the autoradiogram refer to theincrease in miR-18 levels as a multiple of that in RasGfp T1. FIG. 3Cshows immunoblotting analysis of Tsp1 and CTGF expression levels inRasGfpMyc cells transfected with antisense 2′-O-methyloligoribonucleotides targeting components of the miR-17-92 cluster. Theleft hand panel shows cells transfected with mixtures of scrambled ormiR-17-92-specific oligoribonucleotides. Mock-transfected cells wereused as an additional control. The panel on the right shows the samecells transfected with oligoribonucleotides targeting individualmicroRNAs. FIG. 3D shows the results of RT-PCR performed on Ras-onlycolonocytes transduced with either empty vector (RasPuro) or themiR-17-92-encoding retrovirus (RasPuroMIR). PCR primers were specificfor the human miR-17-92 pre-miRNA and did not detect the endogenousmouse transcript. FIG. 3E shows RNA blot analysis of the same cells.RasGfp and RasGfpMyc cells were used for comparison. Other designationsare the same as in FIG. 3B. FIG. 3F shows immunoblotting analysis ofTsp1 and CTGF expression levels in RasPuro versus RasPuroMIR cells.

FIGS. 4A-4E show the effects of miR-17-92 upregulation in Ras-only cellson neoplastic growth. FIG. 4A is a graph showing the results of a cellaccumulation assay performed on RasPuro and RasPuroMIR colonocytes.Numbers of viable cells were assessed using the WST reagent as describedin FIG. 1A. FIG. 4B is a graph showing the average sizes of subcutaneoustumors formed by RasPuro and RasPuroMIR colonocytes in syngeneic mice.“*” indicates statistical significance (P<0.05). P values weredetermined using unpaired Student's t-test. FIG. 4C is a graph showingthe kinetics of tumor formation by RasPuro and RasPuroMIR colonocytesfrom experiment 2 in FIG. 4B. Error bars in 4A-4C represent s.d. FIG. 4Dis a series of four panels showing immunostaining of blood perfusedRasPuro and RasPuroMIR tumors. Immunofluorescent staining corresponds toFITC-conjugated lectin bound to vascular endothelial cells afterintravenous injection. Two independent tumors were assayed.Representative 10× sections from each neoplasm are shown. FIG. 4E is twopanels showing Matrigel neovascularization induced by the same cells asshown in FIG. 4D. The lower image shows richly perfused, large-calibervascular channels that are typical of RasPuroMIR samples.

FIGS. 5A-5C show the interplay between My; miR-17-92, and TSR proteinsin human colon carcinoma cells. FIG. 5A is a Western Blot showing stableknock-down of c-Myc using shRNA. Cells were transduced with a lentiviruscarrying an anti-Myc hairpin from the Sigma MISSION collection. Acontrol shRNA-lentivirus was used as a negative control. The c-Myc,thrombo-spondin-1, and CTGF levels were assessed using Western blotting4 days after infection. Actin was used as a loading control. FIG. 5Bincludes two graphs quantitating the results of real-time RT-PCRanalysis of the miR-17-92 primary transcript. The same cells were testedas in FIG. 5A. Actin-specific PCR product was used as an internalcontrol. FIG. 5C shows immunoflourescence experiments in the left handpanels and immunoblotting analysis in the right hand panels. In theimmunoflourescence experiments, colon carcinoma cells were transfectedwith FITC-labeled oligonucleotides. The immunoblotting analysis showsTsp1 levels in cells transfected with anti-sense 2′-O-methyloligoribonucleotides targeting components of the miR-17-92 cluster.Scrambled oligo and mock-transfected cells were used as negativecontrols.

FIGS. 6A-6B show concerted deregulation of Tsp1 and Myc in human coloncarcinomas and colon cancer cell lines. FIG. 6A shows expression levelsof THBS1 and MYC in colon and, for comparison, CNS tumor cell lines.Individual cell lines are denoted on top. The blue-to-red transitioncorresponds to increasing mRNA levels. FIG. 6B shows down-regulation ofTHBS 1 and upregulation of MYC tnRNAs in human colon carcinoma samples,as compared to surrounding normal mucosas. Data from A. Levine'slaboratory (Notterman et al., 2001) were analyzed using Oncomine 3.0algorithm (available on the world wide web at oncomine.org).

FIG. 7 is a schematic diagram that illustrates the role of Myc in coloncarcinoma neovascularization. Through up-regulation of miR-17-92 andensuing down-regulation of TSR proteins, Myc enhances tumor angiogenesis(“angio”). In this setting, Ki-Ras is chiefly responsible for cellproliferation (“prolif”) although both Ras and p53 contribute to tumorangiogenesis (dotted lines.)

DETAILED DESCRIPTION OF THE INVENTION

The invention generally features methods and compositions for modulatingangiogenesis. In one embodiment, the invention features compositions andmethods featuring inhibitory nucleic acid molecules (e.g., antisenseoligonucleotides) that reduce or eliminate the expression of miRNAswhose expression enhances angiogenesis. Such inhibitory nucleic acidmolecules are useful for the treatment of conditions characterized by anundesirable increase in angiogenesis (e.g., neoplasia, maculardegeneration, diabetic retinopathy, acute inflammation). In particularembodiments, the invention provides compositions for the treatment ofneoplasia, including apoptosis resistant neoplasia, which includestumors that are resistant to chemotherapeutics, and neoplasiacharacterized by a decrease in the expression of one or morethrombospondin type 1 repeat (TSR) proteins (e.g., Connective tissuegrowth factor (CTGF), thrombospondin, type I domain containing 3 isoform3 (THSD3), A disintegrin and metalloproteinase with Tsp motifs 18(ADAMTS18), A disintegrin and metalloproteinase with Tsp motifs 12(ADAMTS12), Thrombospondin 1 (THBS1), Thrombospondin, type 1 domaincontaining 1 (THSD1), A Disintegrin and Metalloproteinase with Tspmotifs 1 (ADAMTS1), A disintegrin and metalloproteinase with Tsp motifs6 (ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3)). In other embodiments,the invention provides compositions featuring polynucleotides comprisingthe nucleic acid sequence of a microRNA of the invention, and methodsfor using such polynucleotides to enhance angiogenesis for the treatmentor prevention of conditions where an increase in angiogenesis wouldprovide a therapeutic effect. Such conditions include, but are notlimited to, ischemia, myocardial infarction, and poorly healing wounds.

MicroRNAs of the mir-17-92 Cluster

MicroRNAs (miRNAs) are 21-23 nucleotide RNA molecules that regulate thestability or translational efficiency of target mRNAs. miRNAs havediverse functions including the regulation of cellular differentiation,proliferation, and apoptosis (Ambros, Nature 431, 350-5 (2004)).Although strict tissue- and developmental-stage-specific expression iscritical for appropriate miRNA function, few mammalian transcriptionfactors that regulate miRNAs have been identified. The proto-oncogenec-MYC encodes a transcription factor that regulates cell proliferation,growth, and apoptosis (Levens, Proc Natl Acad Sci USA 99, 5757-9 (2002).Dysregulated expression or function of c-Myc is one of the most commonabnormalities in human malignancy (Cole et al., Oncogene 18, 2916-24(1999)). c-Myc activated expression of a cluster of six miRNAs on humanchromosome 13.

Inhibitory and Recombinant Nucleic Acid Molecules

As described in more detail below, c-Myc activation of microRNAs of themir-17-92 cluster is associated with angiogenesis. Accordingly, theinvention provides compositions that modulate angiogenesis, as well asmethods of using such compositions for the treatment of diseases wherean increase or decrease in angiogenesis is beneficial. In oneembodiment, the invention provides inhibitory nucleic acid molecules,such as antisense nucleic acid molecules, that decrease the expressionof at least one microRNA of the miR-17-92 cluster. Inhibitory nucleicacid molecules are essentially nucleobase oligomers that may be employedto reduce the expression of a target nucleic acid sequence, such as anucleic acid sequence that encodes a microRNA of the miR-17-92 cluster.The inhibitory nucleic acid molecules provided by the invention includeany nucleic acid molecule sufficient to decrease the expression of anucleic acid molecule of the miR-17-92 cluster by at least 5-10%,desirably by at least 25%-50%, or even by as much as 75%-100%. Each ofthe nucleic acid sequences provided herein may be used, for example, inthe discovery and development of therapeutic antisense nucleic acidmolecules to decrease the expression of a microRNA encoded by themiR-17-92 cluster (e.g., mir-17-5p or mir-20a). If desired, antisensenucleic acid molecules that target one or more microRNAs of themiR-17-92 cluster are administered in combination, such that thecoordinated reduction in the expression of two or more microRNAs encodedby the miR-17-92 cluster is achieved.

The invention is not limited to antisense nucleic acid molecules, butencompasses virtually any single-stranded or double-stranded nucleicacid molecule that decreases expression of a microRNA within themiR-17-92 cluster. The invention further provides catalytic RNAmolecules or ribozymes. Such catalytic RNA molecules can be used toinhibit expression of a microRNA nucleic acid molecule in vivo.

The inclusion of ribozyme sequences within an antisense RNA confersRNA-cleaving activity upon the molecule, thereby increasing the activityof the constructs. The design and use of target RNA-specific ribozymesis described in Haseloff et al., Nature 334:585-591. 1988, and U.S.Patent Application Publication No. 2003/0003469 A1, each of which isincorporated by reference. In various embodiments of this invention, thecatalytic nucleic acid molecule is formed in a hammerhead or hairpinmotif. Examples of such hammerhead motifs are described by Rossi et al.,Nucleic Acids Research and Human Retroviruses, 8:183, 1992. Example ofhairpin motifs are described by Hampel et al., “RNA Catalyst forCleaving Specific RNA Sequences,” filed Sep. 20, 1989, which is acontinuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988,Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al.,Nucleic Acids Research, 18: 299, 1990. These specific motifs are notlimiting in the invention and those skilled in the art will recognizethat all that is important in an enzymatic nucleic acid molecule of thisinvention is that it has a specific substrate binding site which iscomplementary to one or more of the target gene RNA regions, and that ithave nucleotide sequences within or surrounding that substrate bindingsite which impart an RNA cleaving activity to the molecule.

In another approach, the inhibitory nucleic acid molecule is adouble-stranded nucleic acid molecule used for RNA interference(RNAi)-mediated knock-down of the expression of a microRNA. siRNAs arealso useful for the inhibition of microRNAs. See, for example, Nakamotoet al., Hum Mol Genet, 2005. Desirably, the siRNA is designed such thatit provides for the cleavage of a target microRNA of the invention. Inone embodiment, a double-stranded RNA (dsRNA) molecule is made thatincludes between eight and twenty-five (e.g., 8, 10, 12, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25) consecutive nucleobases of a nucleobaseoligomer of the invention. The dsRNA can be two complementary strands ofRNA that have duplexed, or a single RNA strand that has self-duplexed(small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 basepairs, but may be shorter or longer (up to about 29 nucleobases) ifdesired. Double stranded RNA can be made using standard techniques(e.g., chemical synthesis or in vitro transcription). Kits areavailable, for example, from Ambion (Austin, Tex.) and Epicentre(Madison, Wis.). Methods for expressing dsRNA in mammalian cells aredescribed in Brummelkamp et al. Science 296:550-553, 2002; Paddison etal. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol.20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520,2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishiet al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. NatureBiotechnol. 20:500-505 2002, each of which is hereby incorporated byreference. An inhibitory nucleic acid molecule that “corresponds” to amicroRNA of the miR-17-92 cluster comprises at least a fragment of thedouble-stranded gene, such that each strand of the double-strandedinhibitory nucleic acid molecule is capable of binding to thecomplementary strand of the target gene. The inhibitory nucleic acidmolecule need not have perfect correspondence or need not be perfectlycomplementary to the reference sequence. In one embodiment, an siRNA hasat least about 85%, 90%, 95%, 96%, 97%, 98%, or even 99% sequenceidentity with the target nucleic acid or has that degree ofcomplementarity. For example, a 19 base pair duplex or single-strandednucleic acid molecule having 1-2 base pair mismatch is considered usefulin the methods of the invention. In other embodiments, the nucleobasesequence of the inhibitory nucleic acid molecule exhibits 1, 2, 3, 4, 5or more mismatches.

Inhibitory nucleic acid molecules of the invention also include doublestranded nucleic acid “decoys.” Decoy molecules contain a binding sitefor a transcription factor that is responsible for the deregulatedtranscription of a gene of interest. The present invention providesdecoys that competitively block binding to a regulatory element in atarget gene (e.g., miR-17-92 cluster). The competitive inhibition ofc-Myc binding by the decoy results in the indirect inhibition oftranscription of a target microRNA of the miR-17-92 cluster. An overviewof decoy technology is provided by Suda et al., Endocr. Rev., 1999, 20,345-357; S. Yla-Hertttuala and J. F. Martin, The Lancet 355, 213-222,2000). In one therapeutic method, short double-stranded DNA decoymolecules are introduced into cells of a subject. The decoys areprovided in a form that facilitates their entry into target cells of thesubject. Having entered a cell, the decoy specifically binds anendogenous microRNA, thereby competitively inhibiting the microRNA frombinding to an endogenous gene. The decoys are administered in amountsand under conditions whereby binding of the endogenous microRNA to theendogenous gene is effectively competitively inhibited withoutsignificant host toxicity. Depending on the transcription factor, themethods can effect up- or down-regulation of gene expression. Thesubject compositions comprise the decoy molecules in a context thatprovides for pharmacokinetics sufficient for effective therapeutic use.

In other embodiments, the invention provides isolated microRNAs of themiR-17-92 cluster and polynucleotides comprising such sequences. Arecombinant microRNA of the invention may be administered to enhanceangiogenesis in a subject in need thereof. In one approach, the microRNAis administered as a naked RNA molecule. In another approach, it isadministered in an expression vector suitable for expression in amammalian cell.

Another therapeutic approach included in the invention involvesadministration of a recombinant therapeutic, such as a recombinantmicroRNA or an inhibitory nucleic acid molecule, variant, or fragmentthereof, either directly to the site of a potential or actualdisease-affected tissue or systemically (for example, by anyconventional recombinant protein administration technique). The dosageof the administered microRNA depends on a number of factors, includingthe size and health of the individual patient. For any particularsubject, the specific dosage regimes should be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions.

For example, a microRNA or an inhibitory nucleic acid molecule of theinvention may be administered in dosages between about 1 and 100 mg/kg(e.g., 1, 5, 10, 20, 25, 50, 75, and 100 mg/kg). In other embodiments,the dosage ranges from between about 25 and 500 mg/m2/day. Desirably, ahuman patient having a neoplasia receives a dosage between about 50 and300 mg/m2/day (e.g., 50, 75, 100, 125, 150, 175, 200, 250, 275, and300). For the treatment of an opthalmologic disease characterized by anundesired increase in angiogenesis (e.g., neochoroidal vascularization,age-related macular degeneration, diabetic retinopathy), compositions ofthe invention are administered in dosages dosages between about 1 and100 mg/kg (e.g., 1, 5, 10, 20, 25, 50, 75, and 100 mg/kg).

Modified Inhibitory Nucleic Acid Molecules

A desirable inhibitory nucleic acid molecule is one based on 2′-modifiedoligonucleotides containing oligodeoxynucleotide gaps with some or allinternucleotide linkages modified to phosphorothioates for nucleaseresistance. The presence of methylphosphonate modifications increasesthe affinity of the oligonucleotide for its target RNA and thus reducesthe IC50. This modification also increases the nuclease resistance ofthe modified oligonucleotide. It is understood that the methods andreagents of the present invention may be used in conjunction with anytechnologies that may be developed to enhance the stability or efficacyof an inhibitory nucleic acid molecule.

Inhibitory nucleic acid molecules include nucleobase oligomerscontaining modified backbones or non-natural internucleoside linkages.Oligomers having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, modifiedoligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone are also considered to be nucleobase oligomers.Nucleobase oligomers that have modified oligonucleotide backbonesinclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriest-ers, and boranophosphates. Various salts, mixedsalts and free acid forms are also included. Representative UnitedStates patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference.

Nucleobase oligomers having modified oligonucleotide backbones that donot include a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH2 component parts. RepresentativeUnited States patents that teach the preparation of the aboveoligonucleotides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

Nucleobase oligomers may also contain one or more substituted sugarmoieties. Such modifications include 2′-O-methyl and 2′-methoxyethoxymodifications. Another desirable modification is2′-dimethylaminooxyethoxy, 2′-aminopropoxy and 2′-fluoro. Similarmodifications may also be made at other positions on an oligonucleotideor other nucleobase oligomer, particularly the 3′ position of the sugaron the 3′ terminal nucleotide. Nucleobase oligomers may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

In other nucleobase oligomers, both the sugar and the internucleosidelinkage, i.e., the backbone, are replaced with novel groups. Thenucleobase units are maintained for hybridization with a nucleic acidmolecule of the miR-17-92 cluster. Methods for making and using thesenucleobase oligomers are described, for example, in “Peptide NucleicAcids (PNA): Protocols and Applications” Ed. P. E. Nielsen, HorizonPress, Norfolk, United Kingdom, 1999. Representative United Statespatents that teach the preparation of PNAs include, but are not limitedto, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which isherein incorporated by reference. Further teaching of PNA compounds canbe found in Nielsen et al., Science, 1991, 254, 1497-1500.

In other embodiments, a single stranded modified nucleic acid molecule(e.g., a nucleic acid molecule comprising a phosphorothioate backboneand 2′-O-Me sugar modifications is conjugated to cholesterol. Suchconjugated oligomers are known as “antagomirs.” Methods for silencingmicroRNAs in vivo with antagomirs are described, for example, inKrutzfeldt et al., Nature 438: 685-689.

Delivery of Nucleobase Oligomers

Naked oligonucleotides are capable of entering tumor cells or ofentering the stroma and inhibiting the expression or activity of amicroRNA of the miR-17-92 cluster. Nonetheless, it may be desirable toutilize a formulation that aids in the delivery of an inhibitory nucleicacid molecule or other nucleobase oligomers to cells (see, e.g., U.S.Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959,6,346,613, and 6,353,055, each of which is hereby incorporated byreference).

Polynucleotide Therapy

Polynucleotide therapy featuring a polynucleotide encoding a microRNA oran inhibitory nucleic acid molecule or analog thereof that targets amicroRNA of the miR-17-92 cluster is another therapeutic approach formodulating angiogenesis in a subject. Expression vectors encoding themicroRNAs or inhibitory nucleic acid molecules can be delivered to cellsof a subject in need of increased or decreased angiogenesis. The nucleicacid molecules must be delivered to the cells of a subject in a form inwhich they can be taken up and are advantageously expressed so thattherapeutically effective levels can be achieved.

Methods for delivery of the polynucleotides to the cell according to theinvention include using a delivery system such as liposomes, polymers,microspheres, gene therapy vectors, and naked DNA vectors.

Transducing viral (e.g., retroviral, adenoviral, lentiviral andadeno-associated viral) vectors can be used for somatic cell genetherapy, especially because of their high efficiency of infection andstable integration and expression (see, e.g., Cayouette et al., HumanGene Therapy 8:423-430, 1997; Kido et al., Current Eye Research15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649,1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al.,Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, apolynucleotide encoding an inhibitory nucleic acid molecule can becloned into a retroviral vector and expression can be driven from itsendogenous promoter, from the retroviral long terminal repeat, or from apromoter specific for a target cell type of interest. Other viralvectors that can be used include, for example, a vaccinia virus, abovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus(also see, for example, the vectors of Miller, Human Gene Therapy 15-14,1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al.,BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion inBiotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991;Cometta et al., Nucleic Acid Research and Molecular Biology 36:311-322,1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416,1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle etal., Science 259:988-990, 1993; and Johnson, Chest 107:77 S-83S, 1995).Retroviral vectors are particularly well developed and have been used inclinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990;Anderson et al., U.S. Pat. No. 5,399,346).

Non-viral approaches can also be employed for the introduction of aninhibitory nucleic acid molecule therapeutic to a cell of a patientdiagnosed as having a neoplasia. For example, an inhibitory nucleic acidmolecule that targets a microRNA of the miR-17-92 cluster can beintroduced into a cell by administering the nucleic acid in the presenceof lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413,1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am.J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al.,Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal ofBiological Chemistry 264:16985, 1989), or by micro-injection undersurgical conditions (Wolff et al., Science 247:1465, 1990). Preferablythe inhibitory nucleic acid molecules are administered in combinationwith a liposome and protamine.

Gene transfer can also be achieved using non-viral means involvingtransfection in vitro. Such methods include the use of calciumphosphate, DEAF dextran, electroporation, and protoplast fusion.Liposomes can also be potentially beneficial for delivery of DNA into acell.

Inhibitory nucleic acid molecule expression for use in polynucleotidetherapy methods can be directed from any suitable promoter (e.g., thehuman cytomegalovirus (CMV), simian virus 40 (SV40), or metallothioneinpromoters), and regulated by any appropriate mammalian regulatoryelement. For example, if desired, enhancers known to preferentiallydirect gene expression in specific cell types can be used to direct theexpression of a nucleic acid. The enhancers used can include, withoutlimitation, those that are characterized as tissue- or cell-specificenhancers.

For any particular subject, the specific dosage regimes should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions.

Pharmaceutical Compositions

As reported herein, an increase in the expression of microRNAs of themiR-17-92 cluster is associated with an increase in angiogenesis.Accordingly, the invention provides therapeutic compositions thatdecrease the expression of a microRNAs of the miR-17-92 cluster toreduce angiogenesis. In one embodiment, the present invention provides apharmaceutical composition comprising an inhibitory nucleic acidmolecule (e.g., an antisense, siRNA, or shRNA polynucleotide) thatdecreases the expression of one or more nucleic acid molecules encodedby the miR-17-92 cluster (e.g., mir-17-5p or mir-20a). If desired, theinhibitory nucleic acid molecule is administered in combination with achemotherapeutic agent. In another embodiment, a recombinant microRNA ofthe miR-17-92 cluster, or a polynucleotide encoding such a microRNA, isadministered to increase angiogenesis. Polynucleotides of the inventionmay be administered as part of a pharmaceutical composition. Thecompositions should be sterile and contain a therapeutically effectiveamount of the polypeptides or nucleic acid molecules in a unit of weightor volume suitable for administration to a subject.

A recombinant microRNA, an inhibitory nucleic acid molecule of theinvention, or other regulator of a microRNA encoded by the miR-17-92cluster (e.g., mir-17-5p or mir-20a) may be administered within apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form. Conventional pharmaceutical practice may be employed toprovide suitable formulations or compositions to administer thecompounds to patients suffering from a neoplasia (e.g., an apoptosisresistant neoplasia, or a neoplasia characterized by an increase in anangiogenic marker). Administration may begin before the patient issymptomatic. Any appropriate route of administration may be employed,for example, administration may be parenteral, intravenous,intraarterial, subcutaneous, intratumoral, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular,intrathecal, intracisternal, intraperitoneal, intranasal, aerosol,suppository, or oral administration. For example, therapeuticformulations may be in the form of liquid solutions or suspensions; fororal administration, formulations may be in the form of tablets orcapsules; and for intranasal formulations, in the form of powders, nasaldrops, or aerosols.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” Ed. A. R.Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.Formulations for parenteral administration may, for example, containexcipients, sterile water, or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for inhibitory nucleicacid molecules include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Formulationsfor inhalation may contain excipients, for example, lactose, or may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or may be oily solutions foradministration in the form of nasal drops, or as a gel.

The formulations can be administered to human patients intherapeutically effective amounts (e.g., amounts which prevent,eliminate, or reduce a pathological condition) to provide therapy for aneoplastic or ischemic disease or condition. The preferred dosage of anucleobase oligomer of the invention is likely to depend on suchvariables as the type and extent of the disorder, the overall healthstatus of the particular patient, the formulation of the compoundexcipients, and its route of administration.

With respect to a subject having a neoplastic disease or disorder, aneffective amount is sufficient to stabilize, slow, or reduce theproliferation of the neoplasm. Generally, doses of active polynucleotidecompositions of the present invention would be from about 0.01 mg/kg perday to about 1000 mg/kg per day. It is expected that doses ranging fromabout 50 to about 2000 mg/kg will be suitable. Lower doses will resultfrom certain forms of administration, such as intravenousadministration. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits. Multiple dosesper day are contemplated to achieve appropriate systemic levels of anantisense targeting the miR-17-92 cluster (e.g., mir-17-5p or mir-20a).

Therapy

Therapy may be provided wherever cancer therapy is performed: at home,the doctor's office, a clinic, a hospital's outpatient department, or ahospital. Treatment generally begins at a hospital so that the doctorcan observe the therapy's effects closely and make any adjustments thatare needed. The duration of the therapy depends on the kind of neoplasiabeing treated, the age and condition of the patient, the stage and typeof the patient's disease, and how the patient's body responds to thetreatment. Drug administration may be performed at different intervals(e.g., daily, weekly, or monthly). Therapy may be given in on-and-offcycles that include rest periods so that the patient's body has a chanceto build healthy new cells and regain its strength.

Depending on the type of cancer and its stage of development, thetherapy can be used to slow the spreading of the cancer, to slow thecancer's growth, to kill or arrest cancer cells that may have spread toother parts of the body from the original tumor, to relieve symptomscaused by the cancer, or to prevent cancer in the first place. Asdescribed above, if desired, treatment with an inhibitory nucleic acidmolecule of the invention may be combined with therapies for thetreatment of proliferative disease (e.g., radiotherapy, surgery, orchemotherapy). For any of the methods of application described above, aninhibitory nucleic acid molecule of the invention is desirablyadministered intravenously or is applied to the site of neoplasia (e.g.,by injection).

Diagnostics

As described in more detail below, the present invention has identifiedincreases in the expression of microRNAs of the miR-17-92 cluster andc-Myc, and in TSR protein markers that are associated with neoplasia.Neoplasias having increased levels of microRNAs, and/or reduced levelsof a TSR protein, show increased aggressiveness due to enhancedangiogenesis. Thus, alterations in the expression level of one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20) of the following markers is used to diagnose a subject ashaving a particularly aggressive neoplasia: mir-17-5p, mir-18a, mir-19a,mir-20a, mir-19b-1, mir-92-1, c-Myc, E2F1, p21, Connective tissue growthfactor (CTGF), thrombospondin, type I domain containing 3 isoform 3(THSD3), A disintegrin and metalloproteinase with Tsp motifs 18(ADAMTS18), A disintegrin and metalloproteinase with Tsp motifs 12(ADAMTS12), Thrombospondin 1 (THBS1), Thrombospondin, type 1 domaincontaining 1 (THSD1), A disintegrin and metalloproteinase with Tspmotifs 1 (ADAMTS1), a disintegrin and metalloproteinase with Tsp motifs6 (ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3). If desired, alterationsin the expression of one, two, three, four, five, six, seven, eight,nine, ten, eleven, all of these markers is used to diagnose orcharacterize a neoplasia. In another embodiment, the method identifies aneoplasia as amenable to treatment using a method of the invention byassaying an increase in the level of any one or more of the followingangiogenic markers: Connective tissue growth factor (CTGF),thrombospondin, type I domain containing 3 isoform 3 (THSD3), Adisintegrin and metalloproteinase with Tsp motifs 18 (ADAMTS18), Adisintegrin and metalloproteinase with Tsp motifs 12 (ADAMTS12),Thrombospondin 1 (THBS1), Thrombospondin, type 1 domain containing 1(THSD1), A disintegrin and metalloproteinase with Tsp motifs 1(ADAMTS1), a disintegrin and metalloproteinase with Tsp motifs 6(ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3). In one preferredembodiment, the invention characterizes a neoplasia as having anincrease in thrombospondin and at least one additional protein of themiR-17-92 cluster or of an angiogeneic marker.

In one embodiment, a subject is diagnosed as having or having apropensity to develop a neoplasia, the method comprising measuringmarkers in a biological sample from a patient, and detecting analteration in the expression of test marker molecules relative to thesequence or expression of a reference molecule. The markers typicallyinclude a microRNA of the miR-17-92 cluster together with c-Myc or withan angiogenic marker, such as Connective tissue growth factor (CTGF),thrombospondin, type I domain containing 3 isoform 3 (THSD3), Adisintegrin and metalloproteinase with Tsp motifs 18 (ADAMTS18), Adisintegrin and metalloproteinase with Tsp motifs 12 (ADAMTS12),Thrombospondin 1 (THBS1), Thrombospondin, type 1 domain containing 1(THSD1), A disintegrin and metalloproteinase with Tsp motifs 1(ADAMTS1), a disintegrin and metalloproteinase with Tsp motifs 6(ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3). While the followingapproaches describe diagnostic methods featuring a microRNA of themiR-17-92 cluster, the skilled artisan will appreciate that any one ormore of the markers set forth above is useful in such diagnosticmethods.

Increased expression of a microRNA of the miR-17-92 cluster or of a TSRprotein marker is used to identify a neoplasia that is amenable totreatment using a composition or method described herein. Accordingly,the invention provides compositions and methods for identifying suchneoplasias in a subject. Alterations in gene expression are detectedusing methods known to the skilled artisan and described herein. Suchinformation can be used to diagnose a neoplasia or to identify aneoplasia as being amenable to a therapeutic method of the invention.

In one approach, diagnostic methods of the invention are used to assaythe expression of a microRNA of the miR-17-92 cluster or a TSR proteinin a biological sample relative to a reference (e.g., the level ofmicroRNA of the miR-17-92 cluster or TSR present in a correspondingcontrol tissue, such as a healthy tissue or a neoplastic tissue notexhibiting increased angiogenesis). In another approach, diagnosticmethods of the invention are used to assay the expression of a marker.For example, the level of a microRNA of the miR-17-92 cluster isdetected using a nucleic acid probe that specifically binds a microRNAof the miR-17-92 cluster. Exemplary nucleic acid probes thatspecifically bind a microRNA of the miR-17-92 cluster are describedherein. By “nucleic acid probe” is meant any nucleic acid molecule, orfragment thereof, that binds a microRNA encoded by the miR-17-92cluster. Such nucleic acid probes are useful for the diagnosis of aneoplasia.

In one approach, quantitative PCR methods are used to identify anincrease in the expression of a microRNA encoded by the miR-17-92cluster or a TSR protein. In another approach, PCR methods are used toidentify an alteration in the sequence of a microRNA encoded by themiR-17-92 cluster or a TSR protein. The invention provides probes thatare capable of detecting a microRNA encoded by the miR-17-92 cluster ora TSR protein. Such probes may be used to hybridize to a nucleic acidsequence derived from a patient having a neoplasia (e.g., a neoplasiahaving increased angiogenic capacity or apoptosis resistant). Thespecificity of the probe determines whether the probe hybridizes to anaturally occurring sequence, allelic variants, or other relatedsequences. Hybridization techniques may be used to identify mutationsindicative of a neoplasia or may be used to monitor expression levels ofthese genes (for example, by Northern analysis (Ausubel et al., supra).

In yet another embodiment, an immunoassay, radioassay, or otherquantitative assay is used to measure the level of an angiogenic marker,including Connective tissue growth factor (CTGF), thrombospondin, type Idomain containing 3 isoform 3 (THSD3), A disintegrin andmetalloproteinase with Tsp motifs 18 (ADAMTS18), A disintegrin andmetalloproteinase with Tsp motifs 12 (ADAMTS12), Thrombospondin 1(THBS1), Thrombospondin, type 1 domain containing 1 (THSD1), Adisintegrin and metalloproteinase with Tsp motifs 1 (ADAMTS1), adisintegrin and metalloproteinase with Tsp motifs 6 (ADAMTS6), WNT1inducible signaling pathway protein 2 (WISP2), and Brain-specificangiogenesis inhibitor 3 (BAI3). The level of the marker is compared tothe level present in a control sample (e.g., a normal tissue or a tumorsample isolated from a subject having a neoplasia that is notcharacterized by an increase in a mir-17 microRNA and/or an increase inthe level of an angiogenic marker.

In general, the measurement of a nucleic acid molecule or a protein in asubject sample is compared with a diagnostic amount present in areference. A diagnostic amount distinguishes between a neoplastic tissueand a control tissue. The skilled artisan appreciates that theparticular diagnostic amount used can be adjusted to increasesensitivity or specificity of the diagnostic assay depending on thepreference of the diagnostician. In general, any significant increase ordecrease (e.g., at least about 10%, 15%, 30%, 50%, 60%, 75%, 80%, or90%) in the level of test nucleic acid molecule or polypeptide in thesubject sample relative to a reference may be used to diagnose orcharacterize a neoplasia. Test molecules include any one or more ofmir-17-5p, mir-18a, mir-19a, mir-20a, mir-19b-1, mir-92-1, c-Myc, E2F1,p21, Connective tissue growth factor (CTGF), thrombospondin, type Idomain containing 3 isoform 3 (THSD3), A disintegrin andmetalloproteinase with Tsp motifs 18 (ADAMTS18), A disintegrin andmetalloproteinase with Tsp motifs 12 (ADAMTS12), Thrombospondin 1(THBS1), Thrombospondin, type 1 domain containing 1 (THSD1), Adisintegrin and metalloproteinase with Tsp motifs 1 (ADAMTS1), adisintegrin and metalloproteinase with Tsp motifs 6 (ADAMTS6), WNT1inducible signaling pathway protein 2 (WISP2), and Brain-specificangiogenesis inhibitor 3 (BAI3). In one embodiment, the reference is thelevel of test polypeptide or nucleic acid molecule present in a controlsample obtained from a patient that does not have a neoplasia. Inanother embodiment, the reference is a baseline level of test moleculepresent in a biologic sample derived from a patient prior to, during, orafter treatment for a neoplasia. In yet another embodiment, thereference can be a standardized curve.

Types of Biological Samples

The level of markers in a biological sample from a patient having or atrisk for developing a neoplasia can be measured, and an alteration inthe expression of test marker molecule relative to the sequence orexpression of a reference molecule, can be determined in different typesof biologic samples. Test markers include any one or all of thefollowing: mir-17-5p, mir-18a, mir-19a, mir-20a, mir-19b-1, mir-92-1,c-Myc, E2F1, p21, Connective tissue growth factor (CTGF),thrombospondin, type I domain containing 3 isoform 3 (THSD3), Adisintegrin and metalloproteinase with Tsp motifs 18 (ADAMTS18), Adisintegrin and metalloproteinase with Tsp motifs 12 (ADAMTS12),Thrombospondin 1 (THBS1), Thrombospondin, type 1 domain containing 1(THSD1), A disintegrin and metalloproteinase with Tsp motifs 1(ADAMTS1), a disintegrin and metalloproteinase with Tsp motifs 6(ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3). The biological samplesare generally derived from a patient, preferably as a bodily fluid (suchas blood, cerebrospinal fluid, phlegm, saliva, or urine) or tissuesample (e.g. a tissue sample obtained by biopsy).

Kits

The invention provides kits for the diagnosis or monitoring of aneoplasia, such as an apoptosis resistant neoplasia or a neoplasiahaving increased aggressiveness, due to enhanced angiogenesis orangiogenic potential. In one embodiment, the kit detects an alterationin the expression of a Marker (e.g., mir-17-5p, mir-18a, mir-19a,mir-20a, mir-19b-1, mir-92-1, c-Myc, E2F1, p21, Connective tissue growthfactor (CTGF), thrombospondin, type I domain containing 3 isoform 3(THSD3), A disintegrin and metalloproteinase with Tsp motifs 18(ADAMTS18), A disintegrin and metalloproteinase with Tsp motifs 12(ADAMTS12), Thrombospondin 1 (THBS1), Thrombospondin, type 1 domaincontaining 1 (THSD1), A disintegrin and metalloproteinase with Tspmotifs 1 (ADAMTS1), a disintegrin and metalloproteinase with Tsp motifs6 (ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3)) nucleic acid moleculerelative to a reference level of expression. In another embodiment, thekit detects an alteration in the sequence of a miR-17-92 cluster nucleicacid molecule (e.g., a micrRNA of the cluster, such as mir-17-5p,mir-18a, mir-19a, mir-20a, mir-19b-1, mir-92-1) derived from a subjectrelative to a reference sequence. In related embodiments, the kitincludes reagents for monitoring the expression of a miR-17-92 clusternucleic acid molecule or a nucleic acid molecule encoding Connectivetissue growth factor (CTGF), thrombospondin, type I domain containing 3isoform 3 (THSD3), A disintegrin and metalloproteinase with Tsp motifs18 (ADAMTS18), A disintegrin and metalloproteinase with Tsp motifs 12(ADAMTS12), Thrombospondin 1 (THBS1), Thrombospondin, type 1 domaincontaining 1 (THSD1), A disintegrin and metalloproteinase with Tspmotifs 1 (ADAMTS1), a disintegrin and metalloproteinase with Tsp motifs6 (ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3), such as primers orprobes that hybridize to a miR-17-92 cluster nucleic acid molecule.

Optionally, the kit includes directions for monitoring the nucleic acidmolecule levels of a Marker in a biological sample derived from asubject. In other embodiments, the kit comprises a sterile containerwhich contains the primer, probe, antibody, or other detection regents;such containers can be boxes, ampoules, bottles, vials, tubes, bags,pouches, blister-packs, or other suitable container form known in theart. Such containers can be made of plastic, glass, laminated paper,metal foil, or other materials suitable for holding nucleic acids. Theinstructions will generally include information about the use of theprimers or probes described herein and their use in diagnosing aneoplasia. Preferably, the kit further comprises any one or more of thereagents described in the diagnostic assays described herein. In otherembodiments, the instructions include at least one of the following:description of the primer or probe; methods for using the enclosedmaterials for the diagnosis of a neoplasia; precautions; warnings;indications; clinical or research studies; and/or references. Theinstructions may be printed directly on the container (when present), oras a label applied to the container, or as a separate sheet, pamphlet,card, or folder supplied in or with the container.

Patient Monitoring

The disease state or treatment of a patient having a neoplasia can bemonitored using the methods and compositions of the invention. In oneembodiment, the disease state of a patient can be monitored using themethods and compositions of the invention. Such monitoring may beuseful, for example, in assessing the efficacy of a particular drug in apatient. Therapeutics that alter the expression of any one or more ofthe Markers of the invention (e.g., mir-17-5p, mir-18a, mir-19a,mir-20a, mir-19b-1, mir-92-1, c-Myc, E2F1, p21, Connective tissue growthfactor (CTGF), thrombospondin, type I domain containing 3 isoform 3(THSD3), A disintegrin and metalloproteinase with Tsp motifs 18(ADAMTS18), A disintegrin and metalloproteinase with Tsp motifs 12(ADAMTS12), Thrombospondin 1 (THBS1), Thrombospondin, type 1 domaincontaining 1 (THSD1), A disintegrin and metalloproteinase with Tspmotifs 1 (ADAMTS1), a disintegrin and metalloproteinase with Tsp motifs6 (ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3)) are taken asparticularly useful in the invention.

Screening Assays

One embodiment of the invention encompasses a method of identifying anagent that inhibits the expression or activity of a microRNA of themiR-17-92 cluster and/or the expression of a Connective tissue growthfactor (CTGF), thrombospondin, type I domain containing 3 isoform 3(THSD3), A disintegrin and metalloproteinase with Tsp motifs 18(ADAMTS18), A disintegrin and metalloproteinase with Tsp motifs 12(ADAMTS12), Thrombospondin 1 (THBS1), Thrombospondin, type 1 domaincontaining 1 (THSD1), A disintegrin and metalloproteinase with Tspmotifs 1 (ADAMTS1), a disintegrin and metalloproteinase with Tsp motifs6 (ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3). Accordingly, compoundsthat modulate the expression or activity of a miR-17-92 cluster orangiogenic marker nucleic acid molecule, variant, or portion thereof areuseful in the methods of the invention for the treatment or preventionof a neoplasm (e.g., breast, colon, lymph, ovary, stomach, thyroid,testis, and uterine cancer). The method of the invention may measure adecrease in transcription of one or more microRNAs or angiogenic markersof the invention or an alteration in the transcription or translation ofthe target of such a microRNA (e.g., Connective tissue growth factor(CTGF), thrombospondin, type I domain containing 3 isoform 3 (THSD3), Adisintegrin and metalloproteinase with Tsp motifs 18 (ADAMTS18), Adisintegrin and metalloproteinase with Tsp motifs 12 (ADAMTS12),Thrombospondin 1 (THBS1), Thrombospondin, type 1 domain containing 1(THSD1), A disintegrin and metalloproteinase with Tsp motifs 1(ADAMTS1), a disintegrin and metalloproteinase with Tsp motifs 6(ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3), p21, or E2F1). Anynumber of methods are available for carrying out screening assays toidentify such compounds. In one approach, the method comprisescontacting a cell that expresses a microRNA or angiogenic marker with anagent and comparing the level of expression in the cell contacted by theagent with the level of expression in a control cell, wherein an agentthat decreases the expression of a miR-17-92 cluster microRNA expressionor an angiogenic marker thereby inhibits a neoplasia. In anotherapproach, candidate compounds are identified that specifically bind toand alter the activity of a microRNA of the invention. Methods ofassaying such biological activities are known in the art and aredescribed herein. The efficacy of such a candidate compound is dependentupon its ability to interact with a miR-17-92 cluster microRNA orangiogenic marker. Such an interaction can be readily assayed using anynumber of standard binding techniques and functional assays (e.g., thosedescribed in Ausubel et al., supra).

Potential agonists and antagonists of a miR-17-92 cluster microRNA orangiogenic marker include organic molecules, peptides, peptide mimetics,polypeptides, nucleic acid molecules (e.g., double-stranded RNAs,siRNAs, antisense polynucleotides), and antibodies that bind to anucleic acid sequence or polypeptide of the invention and therebyinhibit or extinguish its activity. Potential antagonists also includesmall molecules that bind to the miR-17-92 cluster microRNA therebypreventing binding to cellular molecules with which the microRNAnormally interacts, such that the normal biological activity of themiR-17-92 cluster microRNA is reduced or inhibited. Small molecules ofthe invention preferably have a molecular weight below 2,000 daltons,more preferably between 300 and 1,000 daltons, and still more preferablybetween 400 and 700 daltons. It is preferred that these small moleculesare organic molecules.

Compounds that are identified as binding to a miR-17-92 cluster microRNAor angiogenic marker of the invention with an affinity constant lessthan or equal to 10 mM are considered particularly useful in theinvention. Alternatively, any in vivo protein interaction detectionsystem, for example, any two-hybrid assay may be utilized to identifycompounds that interact with miR-17-92 cluster microRNA or angiogenicmarker. Interacting compounds isolated by this method (or any otherappropriate method) may, if desired, be further purified (e.g., by highperformance liquid chromatography). Compounds isolated by any approachdescribed herein may be used as therapeutics to treat a neoplasia in ahuman patient.

In addition, compounds that inhibit the expression of an miR-17-92cluster microRNA or angiogenic marker whose expression is increased in asubject having a neoplasia are also useful in the methods of theinvention. Any number of methods are available for carrying outscreening assays to identify new candidate compounds that alter theexpression of a miR-17-92 cluster microRNA or angiogenic marker. Theinvention also includes novel compounds identified by theabove-described screening assays. Optionally, such compounds arecharacterized in one or more appropriate animal models to determine theefficacy of the compound for the treatment of a neoplasia. Desirably,characterization in an animal model can also be used to determine thetoxicity, side effects, or mechanism of action of treatment with such acompound. Furthermore, novel compounds identified in any of theabove-described screening assays may be used for the treatment of aneoplasia in a subject. Such compounds are useful alone or incombination with other conventional therapies known in the art.

Test Compounds and Extracts

In general, compounds capable of inhibiting the growth or proliferationof a neoplasia by decreasing the expression or biological activity of amiR-17-92 cluster microRNA (e.g., mir-17-5p or mir-17-20a) or angiogenicmarker are identified from large libraries of either natural product orsynthetic (or semi-synthetic) extracts or chemical libraries accordingto methods known in the art. Methods for making siRNAs are known in theart and are described in the Examples. Numerous methods are alsoavailable for generating random or directed synthesis (e.g.,semi-synthesis or total synthesis) of any number of chemical compounds,including, but not limited to, saccharide-, lipid-, peptide-, andnucleic acid-based compounds. Synthetic compound libraries arecommercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare commercially available from a number of sources, including Biotics(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute(Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.).

In one embodiment, test compounds of the invention are present in anycombinatorial library known in the art, including: biological libraries;peptide libraries (libraries of molecules having the functionalities ofpeptides, but with a novel, non-peptide backbone which are resistant toenzymatic degradation but which nevertheless remain bioactive; see,e.g., Zuckermann, R. N. et al., J. Med. Chem. 37:2678-85, 1994);spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the ‘one-beadone-compound’ library method; and synthetic library methods usingaffinity chromatography selection. The biological library and peptoidlibrary approaches are limited to peptide libraries, while the otherfour approaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, Anticancer Drug Des. 12:145,1997).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al, Proc. Natl. Acad. Sci.U.S.A. 90:6909, 1993; Erb et al, Proc. Natl. Acad. Sci. USA 91:11422,1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho et al, Science261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994; andGallop et al., J. Med. Chem. 37:1233, 1994.

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84,1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S.Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids(Cull at al, Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage(Scott and Smith, Science 249:386-390, 1990; Devlin, Science249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382,1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.).

In addition, those skilled in the art of drug discovery and developmentreadily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their anti-neoplastic activity should beemployed whenever possible.

In an embodiment of the invention, a high thoroughput approach can beused to screen different chemicals for their potency to affect theactivity of a miR-17-92 cluster microRNA (e.g., mir-17-5p or mir-17-20a)or an angiogenic marker.

Those skilled in the field of drug discovery and development willunderstand that the precise source of a compound or test extract is notcritical to the screening procedure(s) of the invention. Accordingly,virtually any number of chemical extracts or compounds can be screenedusing the methods described herein. Examples of such extracts orcompounds include, but are not limited to, plant-, fungal-, prokaryotic-or animal-based extracts, fermentation broths, and synthetic compounds,as well as modification of existing compounds.

When a crude extract is found to alter the biological activity of amiR-17-92 cluster microRNA (e.g., mir-17-5p or mir-17-20a) or angiogenicmarker variant, or fragment thereof, further fractionation of thepositive lead extract is necessary to isolate chemical constituentsresponsible for the observed effect. Thus, the goal of the extraction,fractionation, and purification process is the careful characterizationand identification of a chemical entity within the crude extract havinganti-neoplastic activity. Methods of fractionation and purification ofsuch heterogeneous extracts are known in the art. If desired, compoundsshown to be useful agents for the treatment of a neoplasm are chemicallymodified according to methods known in the art.

The present invention further provides methods of treating diseaseand/or disorders or symptoms thereof which comprise administering atherapeutically effective amount of a pharmaceutical compositioncomprising a compound of the formulae herein to a subject (e.g., amammal such as a human). Thus, one embodiment is a method of treating asubject suffering from or susceptible to a neoplastic disease ordisorder or symptom thereof. The method includes the step ofadministering to the mammal a therapeutic amount of an amount of acompound herein sufficient to treat the disease or disorder or symptomthereof, under conditions such that the disease or disorder is treated.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein, or a composition described herein toproduce such effect. Identifying a subject in need of such treatment canbe in the judgment of a subject or a health care professional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compounds herein, such as a compound of theformulae herein to a subject (e.g., animal, human) in need thereof,including a mammal, particularly a human. Such treatment will besuitably administered to subjects, particularly humans, suffering from,having, susceptible to, or at risk for a disease, disorder, or symptomthereof. Determination of those subjects “at risk” can be made by anyobjective or subjective determination by a diagnostic test or opinion ofa subject or health care provider (e.g., genetic test, enzyme or proteinmarker, Marker (as defined herein), family history, and the like). Thecompounds herein may be also used in the treatment of any otherdisorders in which a neoplasia may be implicated.

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker) (e.g., any target delineated hereinmodulated by a compound herein, a protein or indicator thereof, etc.) ordiagnostic measurement (e.g., screen, assay) in a subject suffering fromor susceptible to a disorder or symptoms thereof associated withneoplasia, in which the subject has been administered a therapeuticamount of a compound herein sufficient to treat the disease or symptomsthereof. The level of Marker determined in the method can be compared toknown levels of Marker in either healthy normal controls or in otherafflicted patients to establish the subject's disease status. Inpreferred embodiments, a second level of Marker in the subject isdetermined at a time point later than the determination of the firstlevel, and the two levels are compared to monitor the course of diseaseor the efficacy of the therapy. In certain preferred embodiments, apre-treatment level of Marker in the subject is determined prior tobeginning treatment according to this invention; this pre-treatmentlevel of Marker can then be compared to the level of Marker in thesubject after the treatment commences, to determine the efficacy of thetreatment.

EXAMPLES Example 1 Myc-Overexpressing Tumors Show RobustNeovascularization

The role of c-Myc in neovascularization of one-hit neoplasms has beenestablished (1-4) and involves both upregulation of pro-angiogenicVEGF4-6 and downregulation of anti-angiogenic Tsp1 (7-9). To address therole of Myc in neovascularization of genetically complex tumors,p53-null mouse colonocytes were used. These cells can be transformed invitro by low-grade over-expression of either activated K-Ras or Myc(10,11). When engrafted into syngeneic mice, subcutaneously ororthotopically (into the cecal wall), Ras-overexpressing cells formedtumors, but their Myc-overexpressing counterparts did not.

Next, MYC was introduced into ras-transformed colonocytes, eitherconstitutively (‘RasGfpMyc’) or in a 4-hydroxytamoxifen (4OHT)-dependentform (‘RasGfpMycER’). No increase in cell accumulation in vitro wasobserved as compared to Ras cells expressing GFP alone (‘RasGfp’) (FIG.1A). Control RasGfp cells formed relatively small tumors. In contrast,RasGfpMyc neoplasms were on average three times larger (FIG. 1B). Thesame increase in tumor sizes was observed with RasGfpMycER cells inanimals continuously treated with 4OHT (FIG. 1B).

To determine the contribution of Myc to neoplastic growth, histologicalexamination of size-matched tumors was carried out. Myc-overexpressingtumors possessed much more robust neovascularization. Especiallynumerous were large-caliber vessels that were richly perfused with redblood cells, as shown in FIG. 1C. Similar differences emerged when thesame sections were stained with lectin to visualize endothelial cells.Whereas RasGfp sections contained only solitary lectin-positive cells,the latter surrounded apparent luminal structures in RasGfpMyc neoplasms(FIG. 1C). In contrast, there was no increase in the density oflymphatic vessels, as judged by staining for the lymphatic-specificLYVE-1 marker (FIG. 1C), in spite of the reported propensity of Myc topromote lymphangiogenesis (5).

Example 2 TSR-Encoding mRNAs are Downregulated by Myc

To determine whether the effects of Myc on angiogenesis are mediated byhypoxia, levels of labile hypoxia-induced factor 1a (HIF 1a) wereassessed in lysates from RasGfp and RasGfpMyc tumors. HIF1a is stableonly under hypoxic conditions and was undetectable in either RasGfp orRasGfpMyc tumors, as shown in FIG. 2A. To determine whetherhypoxia-activated genes are elevated in RasGfpMyc tumors, microarrayanalysis was performed on mRNAs from RasGfp and RasGfpMyc tumors. Therewas no upregulation, at the mRNA level, of a variety of knownhypoxia-activated genes such as Slc2a1 (also known as Glut1) or Vegfa.To investigate possible deregulation of VEGF at the protein level, ELISAwas performed on tumor cell lysates. No difference was observed in VEGFproduction between RasGfp and RasGfpMyc neoplasms (FIG. 2B).

Microarray data was used to analyze the effects of Myc on expression ofother pro- and anti-angiogenic molecules. The list of differentiallyexpressed genes with the Gene Ontology (GO) database was compared todetermine which of 192 known angiogenesis-related genes are subject toregulation by Myc at the mRNA level. No inducers of angiogenesis weresignificantly upregulated. However, the list of Myc-downregulated genesincluded not only thrombospondin-1, but also other proteins withthrombospondin type 1 repeats (TSR): CTGF, spondin-1 (f-spondin),thrombospondin repeat-containing protein 1, clusterin, SPARC, andthrombospondin type I domain-containing protein 6 (See Table 1, below).Table 1 shows TSR-encoding mRNAs downregulated by Myc. In Table 1, “Foldrepression” and “Pvalue” refer to differences in expression levelsbetween the two sets of tumors (RasGfp and RasGfpMyc), per microarraydata. The TSR superfamily members SPARC and spondin-1 are known topossess anti-angiogenic properties (12), whereas CTGF can either promoteor inhibit angiogenesis (13), depending on developmental context (14).

Tsp1 and CTGF data was validated using real-time quantitative RT-PCR(FIG. 2C) and immunoblotting (FIG. 2D). To confirm that theirdownmodulation is directly related to the overexpression of Myc, CTGFand Tsp1 protein levels were examined in RasGfpMycER cells. Treatmentwith 4OHT for 24-72 hours resulted in a reduction of CTGF levels inMycER cell lysates and medium conditioned by pooled MycER clones (FIG.2E). This was also confirmed in several single-cell RasGfpMycER cloneswith detectable CTGF expression. In all such clones, this protein wasdownregulated upon 4OHT treatment (FIG. 2F); one clone (#3) was chosenfor further analyses. Using this clone, it was confirmed that Tsp1 andCTGF are repressed in the presence of activated MycER and return tobasal levels upon subsequent removal of 4OHT (FIG. 2G). Although therepression was less marked than that observed in RasGfpMyc tumors, itindicated that Tsp1 and CTGF are bona fide Myc effectors.

TABLE 1 TSR-encoding mRNAs downregulated by Myc FoldThrombospondin-related proteins repression P-value spondin-1 (f-spondin)8.0 0.05 Thrombospondin 1 7.7 0.006 ADAMTS2 7.6 0.063 WISP2 6.0 0.13Thrombospondin repeat containing [protein] 1 5.7 0.003 Clusterin 5.30.003 CTGF, connective tissue growth factor 5.0 0.001 SPARC (secretedacidic cysteine rich glycoprotein) 3.3 0.1 ADAMTS12 3.1 0.022Thrombospondin type I domain containing 2.3 0.008 [protein] 6

Example 3 Inhibition of miR-17-92 Cluster microRNAs Restored Expressionof Tsp1 and CTGF

It has been previously shown that rather than affecting thethrombospondin-1 promoter, Myc decreases Tsp1 mRNA half-life (8). Morerecently, microRNAs have emerged as important regulators of mRNAstability (15), and at least one microRNA cluster (miR-17-92) isdirectly activated by Myc in human lymphocytes (16) and cooperates withMyc during B-lymphomagenesis (17). Its role in promoting growth of solidtumors has not been fully elucidated. Using quantitative RT-PCRanalysis, it was determined that the steady-state levels of miR-17-92primary transcript were elevated in the presence of overexpressed Myc(FIG. 3A), as were levels of its cleavage products (such as miR-18a), asshown by RNA blotting in FIG. 3B). Notably, several members of the TSRsuperfamily are predicted targets of the miR-17-92 cluster (according tothe MiRanda algorithm (18)). Table 2, shown below, shows TSR proteinsthat are predicted targets of the miR17-92 cluster. Myc-target genes areshown in bold.

TABLE 2 TSR proteins that are predicted targets of the miR17-92 clusterDistance Suppressed Target of from the start P value (per by Myc atwhich and Number of of the 3′ Sanger Inst Gene Gene Description mRNAlevel miRNA hits UTR algorithm) CTGF Connective Yes miR-19a, b 4 1030,1030 0.0015 Tissue growth miR-18 1032, 1033 factor THSD3 thrombospondin,not on array miR-17-5p 4 1170 type I domain miR-20 1171 containing 3miR-18 1172, 1173 isoform 3 ADAMTS18 A disintegrin and No miR-17-5p 4181 metalloproteinase miR-20 183 with Tsp motifs miR-19a, b 235, 236 18ADAMTS12 A disintegrin and Yes miR-19a, b 3 21, 21 metalloproteinasemir-17-3p 26 with Tsp motifs 12 THBS1 Thrombospondin 1 Yes miR-18/19 233, 35 0.0004 (depending on species) THSD1 thrombospondin, not on arraymiR-19a, b 2 78, 76 0.0003 type I domain containing 1 ADAMTS1 Adisintegrin and No miR-20 1 999 0.0007 metalloproteinase with Tsp motifs1 ADAMTS6 A disintegrin and No miR-18 1 192 metalloproteinase with Tspmotifs 6 WISP2 WNT1 inducible Yes miR-17-3p 1 89 0.0003 signalingpathway protein 2 BAI3 Brain-specific Brain- miR-17-3p 1 543angiogenesis specific inhibitor 3

A further question was to determine whether Myc-induced upregulation ofthe miR-17-92 cluster is directly responsible for the downregulation ofTSR proteins in RasGfpMyc cells. As microRNA function can be inhibitedwith specific 2′-O-methyl oligoribonucleotides (19); TSR proteinexpression in transiently transfected RasGfpMyc cells was examined.Using a mixture of antisense oligoribonucleotides targeting sixmicroRNAs from the miR-17-92 cluster, expression of Tsp1 and CTGF inRasGfpMyc cells was partly restored (FIG. 3C, left). Transfection ofantisense oligonucleotides to individual microRNAs further suggestedthat within the cluster, miR-19 is primarily responsible for Tsp1downregulation and miR-18 for CTGF downregulation in response to Myc(FIG. 3C). Antisense oligonucleotides to miR-17-5p (FIG. 3C), miR-20 andmiR-92 did not affect TSR protein levels, consistent with bioinformaticpredictions (see Table 2).

Using retrovirus transduction, Ras cells overexpressing the humanmiR-17-92 cluster (‘RasPuroMIR’, FIG. 3D) were generated, which uponcleavage yield microRNAs that are identical to their mouse counterparts.The level of their overexpression was physiological; that is, comparableto that attained in RasGfpMyc cells (FIG. 3E). As predicted, RasPuroMIRcells produced lower levels of thrombospondin-1 and especially CTGF, ascompared with vector-transduced RasPuro cells (FIG. 3F). In the case ofCTGF, a 90% reduction in mRNA levels was observed, indicative ofregulation at the level of mRNA turnover.

Example 4 miR-17-92 Modulates Tumor Neovascularization

Next, it was examined whether overexpression of miR-17-92 could partlyrecapitulate Myc-induced phenotypes and confer non-cell-autonomousadvantages to RasPuroMIR cells. The water-soluble tetrazolium-1 (WST)assay was used to determine that in vitro RasPuro and RasPuroMIR cellsgrow at a similar rate, as shown in FIG. 4A. However, when cells wereimplanted into C57BL6/NCr mice, RasPuroMIR cells formed tumors that wereon average 1.6-2.5 times larger than RasPuro tumors (data from fourindependent experiments, as shown in FIG. 4B). By monitoring tumorkinetics, it was determined that the two sets of neoplasms initiallygrew at similar rates but diverged when they were several millimeters indiameter, a recognized threshold for angiogenic tumors (20). At thattime point, only RasPuroMIR cells were capable of progressive growth,whereas RasPuro tumors stagnated or even slightly regressed (FIG. 4C,data from experiment 2 in FIG. 4B; similar observations were made inexperiments 1, 3 and 4). To examine the effects of miR-17-92overexpression on tumor vasculature, tumor-bearing mice were injectedintravenously with FITC-conjugated lectin and sacrificed 20 minuteslater. In numerous sections examined using confocal microscopy,RasPuroMIR tumors exhibited a higher density of perfused vessels, asshown in FIG. 4D. Moreover, when the same cells were embedded inMatrigel and injected them subcutaneously into syngeneic hosts,RasPuroMIR cells promoted more vigorous neovascularization, as judged byhemoglobin assay. In particular, only RasPuroMIR implants containedlarge-caliber vascular channels reminiscent of RasGfpMyc tumors (FIG.4E).

Although Myc contributes to angiogenesis in one-hit model neoplasms(1-4), its involvement in angiogenesis is uncertain in the case oftumors (for example, colon carcinomas) in which Myc is coactivated withRas and in which mutations in the TP53 tumor suppressor gene are common.Both activation of Ras and inactivation of p53 are consideredpro-angiogenic. Besides being the repressors of thrombospondin-1, H-Rasand K-Ras are known to upregulate VEGF and increase the activity ofmatrix metalloproteinases (MMP) required for endothelial cell migration(reviewed in ref. 21). The ability of Ras to promote angiogenesis hasbeen documented in a transgenic tumor setting (22). The loss of p53 mayresult in improved stability of hypoxia-induced factor alpha (HIF1a)(23), as well as upregulation of VEGF and downregulation of Tsp1 (24).

In the results presented herein, a combination of mutations in K-ras andTrp53 mutations yielded indolent, poorly vascularized tumors. It was notbefore Myc overexpression and a further decrease in TSR protein levelsthat robust tumor vasculature developed, greatly boosting overallneoplastic growth. Profound downregulation of Tsp1 and CTGF might stemfrom both acute and delayed effects of Myc. In short-term experimentswith MycER-transduced cells, in which only acute effects are assessed,Tsp1 and CTGF were downregulated 65%-80%. Approximately the same levelof repression was apparent in miR-17-92-transduced Ras-cells. Thus,activation of the miR-17-92 cluster can account for the acute effects ofMyc on TSR protein expression. Additional delayed effects could stemfrom the propensity of Myc to activate certain metalloproteinases (25)and thus indirectly affect extracellular proteins.

The data presented herein demonstrates that miR-17-92 can affectnon-cell-autonomous processes, such as tumor neovascularization. Thisindicates that antisense-based microRNA targeting, an emergingtherapeutic technology (19,29), is likely to be effective even againstapoptosis-resistant tumors. It is possible that activation of themiR-17-92 pathway may not be the sole pro-angiogenic event triggered byMyc. However, even partial restoration of TSR expression could tip thebalance between pro- and anti-angiogenic factors in favor of the latter.This shift might afford significant therapeutic benefits in coloncarcinomas, which are known to respond well to anti-angiogenic therapies(30).

Example 5 Interplay Between Myc, miR-17-92, and TSR Proteins Occurs inHuman Colon Cancer Cell Lines

To demonstrate that Myc down-regulates miR-17-92 and TSR proteins inhuman colon cancer cell lines, HCT116.TP53−/− human colon carcinomacells were employed because they bear the same genetic lesions as themurine carcinomas previously used in experiments by the inventors:activated Ras, overexpressed Myc, and loss of p53. The cells wereinfected using lentivirus-encoded anti-Myc hairpins from the SigmaMISSION collection. The efficiency of infection approached 100% (datawith control GFP-encoding lentivirus, not shown). As demonstrated inFIG. 5A, transduction with the hairpin #1377 stably decreases Myc levelsby at least 10-fold in both HCT116.TP53−/− and HCT116.TP53+/+ cells, ascompared to transduction with a control hairpin In HCT116.TP53−/− cells,this leads to upregulation of thrombospondin-1 (FIG. 5A, lanes withinred rectangle). Interestingly, in HCT116-TP53+/+ basal thrombospondin-1levels were higher, consistent with it being a p53 target gene (Dameronet al., 1994). However, these levels didn't increase further upon Mycknockdown. Using the same cells and real-time PCR, it was nextdemonstrated that Myc knockdown also brings about a decrease inmiR-17-92 primary transcript level (FIG. 5B).

To determine whether HCT116.TP53−/− cells are suitable for transienttransfection with antisense 2′-O-methyl oligoribonucleotides againstmiR-17-92, a control FITC-labeled oligonucleotide was used. At least 50%of cells transfected took up Lipofectamine-coated oligonucleotides (FIG.5C, left). Moreover, when a mixture of actual 2′-O-methyloligoribonucleotides was transfected, partial relief of thrombospondin-1repression was observed (FIG. 5C, right). Thus, all three elements ofthe Myc-miR-17-92-TSR “triangle” are present in human cells as well.

These results indicate that repression of TSR proteins is a prerequisitefor neovascularization and aggressive growth of naturally occurringcolon carcinomas. To determine if this is indeed the case, two publiclyavailable microarray databases were analyzed (Cancer Gene Anatomyproject and Oncomine) and searched for patterns of differentialexpression of Myc and TSR proteins. Data from both databases indicatedthat spontaneous human adenocarcinomas have higher levels of Myc andlower levels of thrombospondin-1 mRNAs when compared to normal mucosa(FIG. 6A and FIG. 6B). This is not the case in all neoplastic tissues.For example, thrombospondin-1 is expressed at high levels in linesderived from CNS tumors, where Myc levels are generally low (FIG. 6Aexcerpt from data on “NC1-60” cells lines).

The inverse correlation between Myc and thrombospondin-1 levels innaturally occurring tumors (FIG. 6B) indicates that even in geneticallycomplex colon carcinomas bearing ki-ras and p53 mutations, myc is stillrequired for robust tumor angiogenesis. Furthermore, Myc's contributionto neovasculatization is related to the down-regulation ofthrombospondin-1 and other TSR proteins via post-transcriptionalmechanisms involving up-regulation of the MIR-17-92 microRNA cluster(FIG. 7).

Although Myc contributes to angiogenesis in one-hit model neoplasms(1-4), its involvement in angiogenesis is uncertain in the case oftumors (for example, colon carcinomas) in which Myc is coactivated withRas and in which mutations in the TP53 tumor suppressor gene are common.Both activation of Ras and inactivation of p53 are consideredpro-angiogenic. Besides being the repressors of thrombospondin-1, H-Rasand K-Ras are known to upregulate VEGF and increase the activity ofmatrix metalloproteinases (MMP), which is required for endothelial cellmigration (reviewed in ref. 21). The ability of Ras to promoteangiogenesis has been documented in a transgenic tumor setting (22). Theloss of p53 may result in improved stability of hypoxia-induced factoralpha (HIF1a) (23), as well as upregulation of VEGF and downregulationof Tsp1 (24).

In the results presented herein, a combination of mutations in K-ras andTrp53 mutations yielded indolent, poorly vascularized tumors. It was notbefore Myc overexpression and a further decrease in TSR protein levelsthat robust tumor vasculature developed, greatly boosting overallneoplastic growth. Profound downregulation of Tsp1 and CTGF might stemfrom both acute and delayed effects of Myc. In short-term experimentswith MycER-transduced cells, in which only acute effects are assessed,Tsp1 and CTGF were downregulated 65%-80%. Approximately the same levelof repression was apparent in miR-17-92-transduced Ras-cells. Thus,activation of the miR-17-92 cluster can account for the acute effects ofMyc on TSR protein expression. Additional delayed effects could stemfrom the propensity of Myc to activate certain metalloproteinases (25)and thus indirectly affect extracellular proteins.

The data presented herein demonstrates that miR-17-92 can affectnon-cell-autonomous processes, such as tumor neovascularization. Thisindicates that antisense-based microRNA targeting will be effectiveagainst difficult to treat apoptosis-resistant tumors. Even if theactivation of the miR-17-92 pathway is not the sole pro-angiogenic eventtriggered by Myc, partial restoration of TSR expression is expected toreduce angiogenesis and treat or prevent the onset of colon cancer,including but not limited to pre-malignant colon lesions, such asnon-invasive tumors or polyps.

The above results were obtained using the following methods andmaterials.

Cell Lines and Tumor Production

p53-null colonocytes transformed with retroviruses encoding K-Ras, Mycand MycER have been described previously (10,11). To obtain doublytransduced cells, MigR1 retroviral vectors encoding either Myc or theMyc-estrogen receptor fusion were transfected into GP293 cells usingLipofectamine 2000 (Invitrogen) along with plasmids encoding viralproteins: gag-pol (pGP) and VSV-G protein from vesicular stomatitisvirus. Viral supernatants were harvested 48-72 hours later and added torecipient cells. Polybrene was added to cells to facilitate infection.GFP-positive cells were obtained using FACS.

The mouse stem cell virus-based vector (MSCV) encoding miR-17-92 hasbeen described previously (16). MSCV-miR-17-92-transduced cells wereobtained using puromycin selection. C57BL6/NCr mice were obtained fromthe US National Cancer Institute. Transformed colonocytes were implantedeither subcutaneously or orthotopically, into the wall of the cecum. For4-hydroxytamoxifen via sonication, in corn oil (Sigma) at theconcentration of 10 mg/ml. 4-OHT was administered daily byintraperitoneal injection at the dose of 1 mg per mouse. Cultured cellsexpressing MycER were exposed to 250 nM 4-OHT dissolved in ethanol.

Antisense Inhibition of miR-17-92 Cluster

2′-O-methyl oligoribonucleotides were synthesized by Integrated DNATechnologies (see sequences in Table 3 below). For the analysis of CTGFand Tsp1 protein levels, a mixture of 2′-O-methyl oligoribonucleotides(100 pmol each) targeting individual members of the cluster or 600 pmolof the scrambled oligoribonucleotide were transfected into RasGfpMyccolonocytes growing in six-well dishes (plated at 200,000 cells per well24 h before transfection) using Lipofectamine 2000. Transfectionefficiency (495%) was confirmed using BLOCK-iT Fluorescent Oligo(Invitrogen). Protein lysates were collected 48 hours after transfectionand analyzed by immunoblotting.

TABLE 3Sequences of qRT-PCR primers and 2′-O-methyl oligoribonucleotidesSense oligo Gene qRT-PCR primers Antisense oligo Thrombospondin 1AAGCGCCTATTTACTTCCCACTAG TCCTTTCTTTGACATGCCTGAA CTGFCACCTAAAATCGCCAAGCCTG AGTTCGTGTCCCTTACTTCCTG murine miR-17-ACGCACTTGTTCAGTTCCG TAGTAACCCACCCCCATTCC 92 (primary transcript)humaan miR-17- CTGTCGCCCAATCAAACTG GTCACAATCCCCACCAAAC 92 (primarytranscript) β-actin TTCGTTGCCGGTCCACA ACCAGCGCAGCGATATCG 2′-O-methyloligoribonucleotides miR-17-5p ACUACCUGCACUGUAAGCACUUUG miR-18aUAUCUGCACUAGAUGCAC CUUA miR-19a UCAGUUUUGCAUAGAUUUGCACA miR-19b-1UCAGUUUUGCAUGGAUUUGCACA miR-20a CUACCUGCACUAUAAGCACUUUA miR-92-1CAGGCCGGGACAAGUGCAAUA miR-scrambled AAAACCUUUUGACCGAGCGUGUU

Analyses of Tumor Specimens and Blood Vasculature

Tumor sizes were measured using calipers and tumor weights were recordedon the day of tumor excision. Levels of VEGF in protein extracts weredetermined by ELISA (R&D Systems), using a purified VEGF standard.Alternatively, tissues were fixed in formalin, embedded in paraffin,sectioned and subjected to histological staining orimmunohistochemistry. Vascular endothelial cells were stained using theBandeiraea simplicifolia lectin (BS-1; Sigma). Lymphatics werevisualized using a 1:500 dilution of rabbit polyclonal antibody toLYVE-1 (Research Diagnostics), a biotinylated secondary antibody, andavidin-linked peroxidase. Images were captured at 20× magnificationusing a Nikon Eclipse E600 microscope and a Photometrix CoolSnap camera.

To detect perfused blood vessels, tumor-bearing mice were injectedintravenously with 100 ml of 2 mg/ml FITC-conjugated Lycopersiconesculentum lectin (Vector Labs) 20 minutes before being killed. Afterexcision, tumors were embedded in OCT (Fisher Scientific), frozen inliquid nitrogen, and sectioned into thick (50-mm) sections using acryostat. Slides were examined using an upright Zeiss Axiovert 200Mmicroscope equipped with a Zeiss LSM510 V is/UV META confocal system.FITC fluorescence was detected by a 30 mW argon laser system. Imageswere viewed through a 10× objective, and serial images were acquired at2-mm intervals using LSM510 META V3.2 software. Images were integratedto create a composite projection of a vessel in three dimensions. The invivo Matrigel neovascularization assay has been described in detailearlier (3).

Microarray and microRNA Target Analyses

RNA from RasGfp and RasGfpMyc tumors was used. cDNAs were synthesizedusing in vitro transcription with biotinylated CTP and UTP. LabeledcDNAs were hybridized to the Mouse Genome 430 2.0 Array chip(Affymetrix) using the University of Pennsylvania Microarray Facilitystandard protocol (available on the world wide web atwww.med.upenn.edu/microarr/Data%20Analysis/Affymetrix/methods.htm).Affymetrix MAS5 probe set signals and presence/absence flags werecalculated. The Local Pooled Error (LPE) test for differentialexpression as implemented in S+ArrayAnalyzer v 1.1 (InsightfulCorporation) was applied with 1% Bonferroni multiple testing correctionto median interquartile range-normalized MAS 5 signal values. Theresulting lists were imported into GeneSpring v 6.1 (Silicon Genetics),filtered for Presence (per Affymetrix MAS5 analysis) in two of twosamples in one or more conditions (RasGfp or RasGfpMyc) and thenfiltered for a change of at least 60%.

The comparison with the Gene Ontology database lists was carried outusing DAVID 2.0 software, as implemented at the websiteappsl.niaid.nih.gov/david/. Putative miR-17-92 targets were identifiedusing the MiRanda algorithm18, as implemented at http://www.microrna.organd http://microma.sanger.ac.uk.

Protein and RNA Blotting

For thrombospondin-1 and CTGF expression analysis, either cell lysatesor conditioned medium were used. Myc and HIF 1a expression was detectedin cell lysates. Membranes were probed with antibodies to Tsp1 (Ab-11,Lab Vision), CTGF (L-20, Santa Cruz Biotechnology), Myc (N-262/sc-764,Santa Cruz Biotechnology) and HIF1a (NB 100-105; Novus Biologicals)diluted according to manufacturers' recommendations. Conditioned mediumwas loaded on PAGE without dilution. Appropriate secondary antibodieswere used in horseradish peroxidase-conjugated forms (AmershamBiosciences). Antibody binding was detected using the enhancedchemiluminescence system (Amersham). When indicated, a monoclonalantibody reactive with mouse β-actin (Sigma) was used to confirm equalloading. Detection of mouse miR-17-92 RNAs using RNA blotting wasperformed as described in ref. 16.

Real-Time PCR

Total RNAs were isolated using TRI Reagent (Sigma) and treated with aTURBO DNA-free kit (Ambion). cDNAs were prepared from 2 mg RNA using theSuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Thenucleotide sequences of primers used are provided in Table 1, above.Amplifications were performed using Smartcycler (Cepheid). Typicalconditions were as follows:

95 1C for 150 s (one cycle), then 95 1C for 10 s and 60 1C for 30 s (30cycles). All reactions were performed in duplicates or triplicates toensure accuracy of quantification.

Accession Codes

Microarray data described herein have been deposited to the ArrayExpress(on the world wide web at www.ebi.ac.uk/arrayexpress) database underaccession number E-MEXP-757, which is hereby incorporated by referencein its entirety.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

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What is claimed is:
 1. A method of reducing angiogenesis, the methodcomprising contacting a cell with an effective amount of an inhibitorynucleic acid molecule complementary to at least a portion of a microRNAnucleic acid molecule of the mir-17-92 cluster, thereby reducingangiogenesis.
 2. The method of claim 1, wherein the inhibitory nucleicacid molecule decreases the expression of the microRNA in the cell. 3.The method of claim 1, wherein the cell is present in a tissue or organ.4. The method of claim 1, wherein the cell is a neoplastic cell.
 5. Themethod of claim 1, wherein the cell is an ocular cell.
 6. The method ofclaim 1, wherein the microRNA is selected from the group consisting ofmir-17-5p, mir-18a, mir-19a, mir-20a, mir-19b-1, and mir-92-1.
 7. Themethod of claim 6, wherein microRNA is mir-19.
 8. The method of claim 6,wherein the microRNA is mir-18.
 9. The method of claim 6, wherein thecell is contacted with at least two inhibitory nucleic acid molecules.10. The method of claim 9, wherein the inhibitory nucleic acid moleculesreduce the expression of mir-19 and mir-18.
 11. The method of claim 1,wherein the contact increases expression of a thrombospondin type 1repeat (TSR) protein.
 12. The method of claim 11, wherein the methodincreases expression of Tsp1 or CTGF relative to a reference.
 13. Themethod of claim 12, wherein the reference is the level of Tsp1 or CTGFexpression in the cell prior to treatment or the level present in acorresponding neoplastic control cell.
 14. The method of claim 11,wherein the TSR protein is selected from the group consisting ofthrombospondin type 1 repeats (TSR): spondin-1 (f-spondin),thrombospondin-1, ADAMTS2, WISP2, thrombospondin repeat-containingprotein 1, clusterin, connective tissue growth factor (CTGF), secretedacidic cysteine rich glycoprotein (SPARC), ADAMTS12, thrombospondin typeI domain-containing protein
 6. 15. The method of claim 11, wherein theTSR protein is selected from the group consisting of thrombospondin type1 repeats (TSR): CTGF, THSD3, ADAMTS18, ADAMTS12, THBS1, THSD1, ADAMTS1,ADAMTS6, WISP2, and BAD.
 16. The method of claim 1, wherein theinhibitory nucleic acid molecule is an antisense or siRNA nucleic acidmolecule.
 17. The method of claim 16, wherein the antisense nucleic acidmolecule comprises a nucleobase sequence having at least 95% identity toa sequence selected from the group consisting of: miR-17-5p:ACUACCUGCACuGUAAGCACUUUG; mir-18a: UAUCUGCACUAGAUGCACCUUA; mir-19a:UCAGUUUUGCAUAGAUUUGCACA; mir-19b: UCAGUUUUGCAUGGAUUUGCACA; mir-20a:CUACCUGCACUAUAAGCACUUUA; and mir-92-1: CAGGCCGGGACAAGUGCAAUA.
 18. Amethod for increasing the expression of a TSR protein in a cell, themethod comprising contacting the cell with an effective amount of aninhibitory nucleic acid molecule complementary to at least a portion ofa microRNA nucleic acid molecule of the mir-17-92 cluster, therebyincreasing the expression of a TSR protein.
 19. The method of claim 1,wherein the contact increases expression of a thrombospondin type 1repeat (TSR) protein.
 20. The method of claim 18, wherein the methodincreases expression of Tsp1 or CTGF relative to a reference.
 21. Themethod of claim 20, wherein the reference is the level of Tsp 1 or CTGFexpression in the cell prior to treatment or the level present in acorresponding neoplastic control cell.
 22. The method of claim 18,wherein the inhibitory nucleic acid molecule is an antisense and siRNAnucleic acid molecule.
 23. The method of claim 18, wherein the antisensenucleic acid molecule comprises a nucleobase sequence having at least95% identity to a sequence selected from the group consisting of:miR-17-5p: ACU ACCUGCACuGUAAGC ACUUUG; mir-18a: UAUCUGCACUAGAUGCACCUUA;mir-19a: UCAGUUUUGCAUAGAUUUGCACA; mir-19b: UC AGUUUUGC AUGGAUUUGCACA;mir-20a: CUACCUGCACUAUAAGCACUUUA; and mir-92-1: C AGGCCGGG AC AAGUGCAAUA.
 24. The method of claim 18, wherein the cell is a neoplastic cell.25. The method of claim 18, wherein the cell is an ocular cell.
 26. Amethod of treating an apoptosis resistant neoplasm or chemo-resistantneoplasm in a subject, the method comprising: (a) identifying a subjectas having an apoptosis resistant neoplasm; and (b) administering to thesubject an effective amount of an inhibitory nucleic acid moleculecomplementary to at least a portion of a microRNA of the mir-17-92cluster.
 27. A method of treating or preventing a neoplasm in a subjectin need thereof, the method comprising: (a) identifying a neoplasmhaving an increase in the expression of a TSR protein; and (b)administering to the subject an effective amount of an inhibitorynucleic acid molecule complementary to at least a portion of a microRNAof the mir-17-92 cluster.
 28. A method of treating an ocular diseasecharacterized by increased angiogenesis in a subject, the methodcomprising administering to the subject an effective amount of aninhibitory nucleic acid molecule complementary to at least a portion ofa microRNA of the mir-17-92 cluster.
 29. The method of any one of claims26-29, wherein the ocular disease is macular degeneration, age-relatedmacular degeneration, choroidal neovascularization, or diabeticretinopathy.
 30. The method of claim 29, wherein the method increasesexpression of Tsp1 or CTGF relative to a reference.
 31. The method ofclaim 30, wherein the reference is the level of Tsp1 or CTGF expressionin the cell prior to treatment or the level present in a correspondingneoplastic control cell.
 32. The method of any one of claims 26-29,wherein the inhibitory nucleic acid molecule is an antisense and siRNAnucleic acid molecule.
 33. The method of any one of claims 26-29,wherein the cell is contacted with at least two inhibitory nucleic acidmolecules.
 34. The method of claim 33, wherein the inhibitory nucleicacid molecules reduce the expression of mir-19 and mir-18.
 35. Themethod of any one of claims 26-29, wherein the one or more inhibitorynucleic acid molecules are administered concurrently or within 14 daysof each other in amounts sufficient to inhibit the growth of theapoptosis resistant neoplasm.
 36. The method of claim 26 or 27, whereinthe cancer is selected from the group consisting of lung cancer, breastcancer, cervical cancer, colon cancer, gastric cancer, kidney cancer,leukemia, liver cancer, lymphoma, ovarian cancer, pancreatic cancer,prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer,and uterine cancer
 37. The method of any one of claims 1-36, wherein theinhibitory nucleic acid molecule is administered at in an amount betweenabout 100 to 300 mg/m/day.
 38. The method of any one of claims 1-36,wherein the inhibitory nucleic acid molecule is administeredsystemically to a subject.
 39. The method of any one of claims 1-36,wherein the method targets the tumor and the tumor microenvironment. 40.The method of claim 39, wherein the tumor microenvironment comprisesstromal cells, fibroblasts, endothelial cells, inflammatory cells,smooth muscle cells, and pericytes.
 41. A method of characterizing atumor as amenable to treatment with the method of any one of claim 1-40,the method comprising assaying the expression of a microRNA encoded bythe miR-17-92 cluster.
 42. A method of characterizing a tumor asamenable to treatment with the method of any one of claim 1-40, themethod comprising assaying the expression of a TSR protein.
 43. Themethod of claim 41 or 42, wherein the method detects an increase or adecrease in the expression of a TSR protein relative to a reference. 44.A method of selecting a treatment for a subject having a neoplasm, themethod comprising (a) detecting an increase the expression of a microRNAencoded by a mir-17-92 cluster or a TSR protein; and (b) identifying thepatient as having a neoplasm amenable to treatment with an inhibitorynucleic acid molecule that reduces the expression of a microRNA of themiR-17-92 cluster.
 45. The method of claim 44, wherein the methoddetects an increase in the expression of a microRNA and a TSR protein.46. A method of monitoring the treatment of a subject having aneoplasia, the method comprising: (a) assaying the expression of a TSRprotein in a cell of the subject; and (b) detecting an increase or adecrease in the expression of a TSR protein relative to a reference. 47.The method of claim 46, wherein an increase in the expression of the TSRprotein indicates that the treatment is beneficial.
 48. An isolatednucleic acid molecule having at least 85% nucleic acid sequence identityto a microRNA encoded by the miR-17-92 cluster, wherein expression ofthe nucleic acid molecule in a cell enhances angiogenesis.
 49. Thenucleic acid molecule of claim 48, wherein the nucleic acid molecule hasat least 85% sequence identity to (human) a microRNA selected from thegroup consisting of miR-17-92, miR-19, and miR-18.
 50. The nucleic acidmolecule of any one of claims 48-49, wherein the nucleic acid moleculecomprises at least one modification.
 51. The nucleic acid molecule ofclaim 50, wherein the modification is a non-natural internucleotidelinkage, modified backbone, substituted sugar moiety, or cholesterolconjugation.
 52. An expression vector encoding a nucleic acid moleculeof any one of claims 48-49.
 53. The vector of claim 52, wherein thevector is a retroviral, adenoviral, adeno-associated viral, orlentiviral vector.
 54. The vector of claim 53, wherein the vectorcomprises a promoter suitable for expression in a mammalian cell,wherein the promoter is operably linked to the nucleic acid molecule.55. A cell comprising the vector of claim 5 or a nucleic acid moleculeof any one of claims 48-49.
 56. The cell of claim 55, wherein the cellis in vivo.
 57. The cell of claim 56, wherein the cell is a neoplasticcolonocyte cell in vivo.
 58. The cell of claim 8, wherein the cell isapoptosis-resistant.
 59. A method of enhancing angiogenesis, the methodcomprising contacting the cell with an effective amount of a nucleicacid molecule comprising at least a portion of a microRNA nucleic acidmolecule of the mir-17-92 cluster.
 60. The method of claim 20, whereinthe cell further expresses one or more of a thrombospondin familyprotein.
 61. A method of identifying an agent that reduces angiogenesis,the method comprising (a) contacting a cell that expresses a TSRselected from the group consisting of: Connective tissue growth factor(CTGF), thrombospondin, type I domain containing 3 isoform 3 (THSD3), Adisintegrin and metalloproteinase with Tsp motifs 18 (ADAMTS18), Adisintegrin and metalloproteinase with Tsp motifs 12 (ADAMTS12),Thrombospondin 1 (THBS1), Thrombospondin, type 1 domain containing 1(THSD1), A disintegrin and metalloproteinase with Tsp motifs 1(ADAMTS1), A disintegrin and metalloproteinase with Tsp motifs 6(ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAD) and a microRNA of themir-17-92 cluster with a test agent; and (b) detecting an increase inthe level of TSR expression in the cell contacted by the agent with thelevel present in a control cell, wherein the increase in TSR expressionidentifying the agent as reducing angiogenesis.
 62. A method ofidentifying an agent that treats or prevents an apoptosis resistantneoplasm, the method comprising (a) contacting a cell that expresses amicroRNA of the mir-17-92 cluster with an agent, and (b) detecting areduction in the level of microRNA expression in the cell contacted bythe agent with the level of expression in a control cell, wherein anagent that decreases microRNA expression thereby treats or prevents aneoplasm.
 63. The method of claim 62, wherein the cell further expressesreduced levels of a TSR protein selected from the group consisting of:Connective tissue growth factor (CTGF), thrombospondin, type I domaincontaining 3 isoform 3 (THSD3), A disintegrin and metalloproteinase withTsp motifs 18 (ADAMTS18), A disintegrin and metalloproteinase with Tspmotifs 12 (ADAMTS 12), Thrombospondin 1 (THBS1), Thrombospondin, type 1domain containing 1 (THSD1), A disintegrin and metalloproteinase withTsp motifs 1 (ADAMTS1), A disintegrin and metalloproteinase with Tspmotifs 6 (ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2),and Brain-specific angiogenesis inhibitor 3 (BAI3) relative to areference cell.
 64. The method of claim 33, wherein the decrease inexpression is by at least about 5%.
 65. A method for diagnosing asubject as having or having a propensity to develop an apoptosisresistant neoplasia, the method comprising (a) measuring the level of aTSR protein selected from the group consisting of: Connective tissuegrowth factor (CTGF), thrombospondin, type I domain containing 3 isoform3 (THSD3), A disintegrin and metalloproteinase with Tsp motifs 18(ADAMTS18), A disintegrin and metalloproteinase with Tsp motifs 12(ADAMTS12), Thrombospondin 1 (THBS1), Thrombospondin, type 1 domaincontaining 1 (THSD1), A disintegrin and metalloproteinase with Tspmotifs 1 (ADAMTS1), A disintegrin and metalloproteinase with Tsp motifs6 (ADAMTS6), WNT1 inducible signaling pathway protein 2 (WISP2), andBrain-specific angiogenesis inhibitor 3 (BAI3) in a biological samplefrom the subject; and (b) comparing the level of the TSR protein in thesubject to the level present in a control subject, wherein a reducedlevel of TSR protein indicates the subject has or has a propensity todevelop an apoptosis resistant neoplasia.
 66. A method for diagnosing asubject as having or having a propensity to develop an apoptosisresistant neoplasia, the method comprising (a) measuring the level of amir17-92 encoded microRNA in a biological sample derived from a subject;and (b) detecting an increased level of the microRNA relative to thelevel present in a control sample, wherein a, wherein an increase in thelevel of the mir-17-92 encoded microRNA indicates the subject has or hasa propensity to develop a an apoptosis resistant neoplasia.
 67. Themethod of claim 65 or 66, wherein the level of mir-17-92 is detected ina microarray assay, an immunoassay, or a radioassay.
 68. The method ofclaim 65 or 66, wherein the method comprises measuring the level ofnucleic acid molecule or polypeptide.
 69. A pharmaceutical compositionfor treating an apoptosis resistant neoplasm in a subject comprising aneffective amount of an inhibitory nucleic acid molecule that iscomplementary to at least a fragment of mir-17-92 in a pharmaceuticallyacceptable excipient.